Nucleic acid-polypeptide compositions and methods of inducing exon skipping

ABSTRACT

Disclosed herein are molecules and pharmaceutical compositions that induce an insertion, deletion, duplication, or alteration in an incorrectly spliced mRNA transcript to induce exon skipping or exon inclusion. Also described herein include methods for treating a disease or disorder that comprises a molecule or a pharmaceutical composition that induces an insertion, deletion, duplication, or alteration in an incorrectly spliced mRNA transcript to induce exon skipping or exon inclusion.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.16/129,696, filed Sep. 12, 2018, which is a continuation of U.S.application Ser. No. 16/128,450 filed Sep. 11, 2018, which is acontinuation of the International Application No. PCT/US2018/012672,filed Jan. 5, 2018, which claims the benefit of U.S. Provisional PatentApplication No. 62/561,939 filed on Sep. 22, 2017 and 62/443,514 filedon Jan. 6, 2017, each of which is incorporated herein by reference inits entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 12, 2018, isnamed 45532-715_303_SL.txt and is 210,679 bytes in size.

BACKGROUND OF THE DISCLOSURE

Modulation of RNA function is a developing area of therapeutic interest.Drugs that affect mRNA stability like antisense oligonucleotides andshort interfering RNAs are one way to modulate RNA function. Anothergroup of oligonucleotides can modulate RNA function by altering theprocessing of pre-mRNA to include or exclude specific regions ofpre-mRNAs from the ultimate gene product: the encoded protein. As such,oligonucleotide therapeutics represent a means of modulating proteinexpression in disease states and as such have utility as therapeutics.

SUMMARY OF THE DISCLOSURE

Disclosed herein, in certain embodiments, are molecules andpharmaceutical compositions for modulating RNA processing.

Disclosed herein, in certain embodiments, are methods of treating adisease or disorder caused by an incorrectly spliced mRNA transcript ina subject in need thereof, the method comprising: administering to thesubject a polynucleic acid molecule conjugate; wherein the polynucleicacid molecule conjugate is conjugated to a cell targeting bindingmoiety; wherein the polynucleotide optionally comprises at least one 2′modified nucleotide, at least one modified internucleotide linkage, orat least one inverted abasic moiety; wherein the polynucleic acidmolecule conjugate induces insertion, deletion, duplication, oralteration in the incorrectly spliced mRNA transcript to induce exonskipping or exon inclusion in the incorrectly spliced mRNA transcript togenerate a fully processed mRNA transcript; and wherein the fullyprocessed mRNA transcript encodes a functional protein, thereby treatingthe disease or disorder in the subject. In some embodiments, the diseaseor disorder is further characterized by one or more mutations in themRNA. In some embodiments, the disease or disorder comprises aneuromuscular disease, a genetic disease, cancer, a hereditary disease,or a cardiovascular disease. In some embodiments, the disease ordisorder is muscular dystrophy. In some embodiments, the disease ordisorder is Duchenne muscular dystrophy. In some embodiments, the exonskipping is of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of theDMD gene. In some embodiments, the exon skipping is of exon 23 of theDMD gene. In some embodiments, the polynucleic acid molecule conjugatecomprises a structure of Formula (I):A-X—B   Formula I

-   -   wherein,    -   A comprises a binding moiety;    -   B consists of a polynucleotide; and    -   X consists of a bond or first linker.        In some embodiments, the polynucleic acid molecule conjugate        comprises a structure of Formula (II):        A-X—B—Y—C   Formula II    -   wherein,    -   A comprises a binding moiety;    -   B consists of a polynucleotide;    -   C consists of a polymer;    -   X consists of a bond or first linker; and    -   Y consists of a bond or second linker.        In some embodiments, the polynucleic acid molecule conjugate        comprises a structure of Formula (III):        A-X—C—Y—B   Formula III    -   wherein,    -   A comprises a binding moiety;    -   B consists of a polynucleotide;    -   C consists of a polymer;    -   X consists of a bond or first linker; and    -   Y consists of a bond or second linker.        In some embodiments, the at least one 2′ modified nucleotide        comprises a morpholino, 2′-O-methyl, 2′-O-methoxyethyl        (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro,        2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl        (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),        T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or        2′-O—N-methylacetamido (2′-O-NMA) modified nucleotide. In some        embodiments, the at least one 2′ modified nucleotide comprises        locked nucleic acid (LNA), ethylene nucleic acid (ENA), or a        peptide nucleic acid (PNA). In some embodiments, the at least        one 2′ modified nucleotide comprises a morpholino. In some        embodiments, the at least one inverted basic moiety is at least        one terminus. In some embodiments, the at least one modified        internucleotide linkage comprises a phosphorothioate linkage or        a phosphorodithioate linkage. In some embodiments, the        polynucleic acid molecule is at least from about 10 to about 30        nucleotides in length. In some embodiments, the polynucleic acid        molecule is at least one of: from about 15 to about 30, from        about 18 to about 25, from about 18 to about 24, from about 19        to about 23, or from about 20 to about 22 nucleotides in length.        In some embodiments, the polynucleic acid molecule is at least        about 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in        length. In some embodiments, the polynucleic acid molecule        comprises at least one of: from about 5% to about 100%        modification, from about 10% to about 100% modification, from        about 20% to about 100% modification, from about 30% to about        100% modification, from about 40% to about 100% modification,        from about 50% to about 100% modification, from about 60% to        about 100% modification, from about 70% to about 100%        modification, from about 80% to about 100% modification, and        from about 90% to about 100% modification. In some embodiments,        the polynucleic acid molecule comprises at least one of: from        about 10% to about 90% modification, from about 20% to about 90%        modification, from about 30% to about 90% modification, from        about 40% to about 90% modification, from about 50% to about 90%        modification, from about 60% to about 90% modification, from        about 70% to about 90% modification, and from about 80% to about        100% modification. In some embodiments, the polynucleic acid        molecule comprises at least one of: from about 10% to about 80%        modification, from about 20% to about 80% modification, from        about 30% to about 80% modification, from about 40% to about 80%        modification, from about 50% to about 80% modification, from        about 60% to about 80% modification, and from about 70% to about        80% modification. In some embodiments, the polynucleic acid        molecule comprises at least one of: from about 10% to about 70%        modification, from about 20% to about 70% modification, from        about 30% to about 70% modification, from about 40% to about 70%        modification, from about 50% to about 70% modification, and from        about 60% to about 70% modification. In some embodiments, the        polynucleic acid molecule comprises at least one of: from about        10% to about 60% modification, from about 20% to about 60%        modification, from about 30% to about 60% modification, from        about 40% to about 60% modification, and from about 50% to about        60% modification. In some embodiments, the polynucleic acid        molecule comprises at least one of: from about 10% to about 50%        modification, from about 20% to about 50% modification, from        about 30% to about 50% modification, and from about 40% to about        50% modification. In some embodiments, the polynucleic acid        molecule comprises at least one of: from about 10% to about 40%        modification, from about 20% to about 40% modification, and from        about 30% to about 40% modification. In some embodiments, the        polynucleic acid molecule comprises at least one of: from about        10% to about 30% modification, and from about 20% to about 30%        modification. In some embodiments, the polynucleic acid molecule        comprises from about 10% to about 20% modification. In some        embodiments, the polynucleic acid molecule comprises from about        15% to about 90%, from about 20% to about 80%, from about 30% to        about 70%, or from about 40% to about 60% modifications. In some        embodiments, the polynucleic acid molecule comprises at least        about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%        modification. In some embodiments, the polynucleic acid molecule        comprises at least about 3, about 4, about 5, about 6, about 7,        about 8, about 9, about 10, about 11, about 12, about 13, about        14, about 15, about 16, about 17, about 18, about 19, about 20,        about 21, about 22 or more modifications. In some embodiments,        the polynucleic acid molecule comprises at least about 1, about        2, about 3, about 4, about 5, about 6, about 7, about 8, about        9, about 10, about 11, about 12, about 13, about 14, about 15,        about 16, about 17, about 18, about 19, about 20, about 21,        about 22 or more modified nucleotides. In some embodiments, the        polynucleic acid molecule comprises a single strand. In some        embodiments, the polynucleic acid molecule comprises two or more        strands. In some embodiments, the polynucleic acid molecule        comprises a first polynucleotide and a second polynucleotide        hybridized to the first polynucleotide to form a double-stranded        polynucleic acid molecule. In some embodiments, the second        polynucleotide comprises at least one modification. In some        embodiments, the first polynucleotide and the second        polynucleotide are RNA molecules. In some embodiments, the first        polynucleotide and the second polynucleotide are siRNA        molecules. In some embodiments, X and Y are independently a        bond, a degradable linker, a non-degradable linker, a cleavable        linker, or a non-polymeric linker group. In some embodiments, X        is a bond. In some embodiments, X is a C₁-C₆ alkyl group. In        some embodiments, Y is a C₁-C₆ alkyl group. In some embodiments,        X is a homobifunctional linker or a heterobifunctional linker,        optionally conjugated to a C₁-C₆ alkyl group. In some        embodiments, Y is a homobifunctional linker or a        heterobifunctional linker. In some embodiments, the binding        moiety is an antibody or binding fragment thereof. In some        embodiments, the antibody or binding fragment thereof comprises        a humanized antibody or binding fragment thereof, chimeric        antibody or binding fragment thereof, monoclonal antibody or        binding fragment thereof, monovalent Fab′, divalent Fab2,        single-chain variable fragment (scFv), diabody, minibody,        nanobody, single-domain antibody (sdAb), or camelid antibody or        binding fragment thereof. In some embodiments, C is polyethylene        glycol. In some embodiments, C has a molecular weight of about        5000 Da. In some embodiments, A-X is conjugated to the 5′ end of        B and Y—C is conjugated to the 3′ end of B. In some embodiments,        Y—C is conjugated to the 5′ end of B and A-X is conjugated to        the 3′ end of B. In some embodiments, A-X, Y—C or a combination        thereof is conjugated to an internucleotide linkage group. In        some embodiments, methods further comprise D. In some        embodiments, D is conjugated to C or to A. In some embodiments,        D is conjugated to the molecule conjugate of Formula (II)        according to Formula (IV):        (A-X—B—Y—C_(c))L-D   Formula IV    -   wherein,    -   A comprises a binding moiety;    -   B consists of a polynucleotide;    -   C consists of a polymer;    -   X consists of a bond or first linker;    -   Y consists of a bond or second linker;    -   L consists of a bond or third linker;    -   D consists of an endosomolytic moiety; and    -   c is an integer between 0 and 1; and    -   wherein the polynucleotide comprises at least one 2′ modified        nucleotide, at least one modified internucleotide linkage, or an        inverted abasic moiety; and D is conjugated anywhere on A, B, or        C.        In some embodiments, D is INF7 or melittin. In some embodiments,        L is a C₁-C₆ alkyl group. In some embodiments, L is a        homobifunctional linker or a heterobifunctional linker. In some        embodiments, methods further comprise at least a second binding        moiety A. In some embodiments, the at least second binding        moiety A is conjugated to A, to B, or to C.

Disclosed herein, in some embodiments, are methods of inducing aninsertion, deletion, duplication, or alteration in the incorrectlyspliced mRNA transcript to induce exon skipping or exon inclusion in theincorrectly spliced mRNA transcript, the method comprising: contacting atarget cell with a polynucleic acid molecule conjugate, wherein thepolynucleotide comprises at least one 2′ modified nucleotide, at leastone modified internucleotide linkage, or at least one inverted abasicmoiety; hybridizing the polynucleic acid molecule conjugate to theincorrectly spliced mRNA transcript within the target cell to induce aninsertion, deletion, duplication, or alteration in the incorrectlyspliced mRNA transcript to induce exon skipping or exon inclusion,wherein the incorrectly spliced mRNA transcript is capable of encoding afunctional form of a protein; and translating the functional form of aprotein from a fully processed mRNA transcript of the previous step. Insome embodiments, the target cell is a target cell of a subject. In someembodiments, the incorrectly spliced mRNA transcript further induces adisease or disorder. In some embodiments, the disease or disorder isfurther characterized by one or more mutations in the mRNA. In someembodiments, the disease or disorder comprises a neuromuscular disease,a genetic disease, cancer, a hereditary disease, or a cardiovasculardisease. In some embodiments, the disease or disorder is musculardystrophy. In some embodiments, the disease or disorder is Duchennemuscular dystrophy. In some embodiments, the exon skipping is of exon 8,23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In someembodiments, the exon skipping is of exon 23 of the DMD gene. In someembodiments, the polynucleic acid molecule conjugate comprises astructure of Formula (I):A-X—B   Formula I

-   -   wherein,    -   A comprises a binding moiety;    -   B consists of a polynucleotide; and    -   X consists of a bond or first linker.        In some embodiments, the polynucleic acid molecule conjugate        comprises a structure of Formula (II):        A-X—B—Y—C   Formula II    -   wherein,    -   A comprises a binding moiety;    -   B consists of a polynucleotide;    -   C consists of a polymer;    -   X consists of a bond or first linker; and    -   Y consists of a bond or second linker.        In some embodiments, the polynucleic acid molecule conjugate        comprises a structure of Formula (III):        A-X—C—Y—B   Formula III    -   wherein,    -   A comprises a binding moiety;    -   B consists of a polynucleotide;    -   C consists of a polymer;    -   X consists of a bond or first linker; and    -   Y consists of a bond or second linker.        In some embodiments, the at least one 2′ modified nucleotide        comprises a morpholino, 2′-O-methyl, 2′-O-methoxyethyl        (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro,        2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl        (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),        T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or        2′-O—N-methylacetamido (2′-O-NMA) modified nucleotide. In some        embodiments, the at least one 2′ modified nucleotide comprises        locked nucleic acid (LNA), ethylene nucleic acid (ENA), peptide        nucleic acid (PNA). In some embodiments, the at least one 2′        modified nucleotide comprises a morpholino. In some embodiments,        the at least one inverted basic moiety is at least one terminus.        In some embodiments, the at least one modified internucleotide        linkage comprises a phosphorothioate linkage or a        phosphorodithioate linkage. In some embodiments, the polynucleic        acid molecule is at least from about 10 to about 30 nucleotides        in length. In some embodiments, the polynucleic acid molecule is        at least one of: from about 15 to about 30, from about 18 to        about 25, from about 18 to about 24, from about 19 to about 23,        or from about 20 to about 22 nucleotides in length. In some        embodiments, the polynucleic acid molecule is at least about 16,        17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In        some embodiments, the polynucleic acid molecule comprises at        least one of: from about 5% to about 100% modification, from        about 10% to about 100% modification, from about 20% to about        100% modification, from about 30% to about 100% modification,        from about 40% to about 100% modification, from about 50% to        about 100% modification, from about 60% to about 100%        modification, from about 70% to about 100% modification, from        about 80% to about 100% modification, and from about 90% to        about 100% modification. In some embodiments, the polynucleic        acid molecule comprises at least one of: from about 10% to about        90% modification, from about 20% to about 90% modification, from        about 30% to about 90% modification, from about 40% to about 90%        modification, from about 50% to about 90% modification, from        about 60% to about 90% modification, from about 70% to about 90%        modification, and from about 80% to about 100% modification. In        some embodiments, the polynucleic acid molecule comprises at        least one of: from about 10% to about 80% modification, from        about 20% to about 80% modification, from about 30% to about 80%        modification, from about 40% to about 80% modification, from        about 50% to about 80% modification, from about 60% to about 80%        modification, and from about 70% to about 80% modification. In        some embodiments, the polynucleic acid molecule comprises at        least one of: from about 10% to about 70% modification, from        about 20% to about 70% modification, from about 30% to about 70%        modification, from about 40% to about 70% modification, from        about 50% to about 70% modification, and from about 60% to about        70% modification. In some embodiments, the polynucleic acid        molecule comprises at least one of: from about 10% to about 60%        modification, from about 20% to about 60% modification, from        about 30% to about 60% modification, from about 40% to about 60%        modification, and from about 50% to about 60% modification. In        some embodiments, the polynucleic acid molecule comprises at        least one of: from about 10% to about 50% modification, from        about 20% to about 50% modification, from about 30% to about 50%        modification, and from about 40% to about 50% modification. In        some embodiments, the polynucleic acid molecule comprises at        least one of: from about 10% to about 40% modification, from        about 20% to about 40% modification, and from about 30% to about        40% modification. In some embodiments, the polynucleic acid        molecule comprises at least one of: from about 10% to about 30%        modification, and from about 20% to about 30% modification. In        some embodiments, the polynucleic acid molecule comprises from        about 10% to about 20% modification. In some embodiments, the        polynucleic acid molecule comprises from about 15% to about 90%,        from about 20% to about 80%, from about 30% to about 70%, or        from about 40% to about 60% modifications. In some embodiments,        the polynucleic acid molecule comprises at least about 15%, 20%,        30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modification. In        some embodiments, the polynucleic acid molecule comprises at        least about 3, about 4, about 5, about 6, about 7, about 8,        about 9, about 10, about 11, about 12, about 13, about 14, about        15, about 16, about 17, about 18, about 19, about 20, about 21,        about 22 or more modifications. In some embodiments, the        polynucleic acid molecule comprises at least about 1, about 2,        about 3, about 4, about 5, about 6, about 7, about 8, about 9,        about 10, about 11, about 12, about 13, about 14, about 15,        about 16, about 17, about 18, about 19, about 20, about 21,        about 22 or more modified nucleotides. In some embodiments, the        polynucleic acid molecule comprises a single strand. In some        embodiments, the polynucleic acid molecule comprises two or more        strands. In some embodiments, the polynucleic acid molecule        comprises a first polynucleotide and a second polynucleotide        hybridized to the first polynucleotide to form a double-stranded        polynucleic acid molecule. In some embodiments, the second        polynucleotide comprises at least one modification. In some        embodiments, the first polynucleotide and the second        polynucleotide are RNA molecules. In some embodiments, the first        polynucleotide and the second polynucleotide are siRNA        molecules. In some embodiments, X and Y are independently a        bond, a degradable linker, a non-degradable linker, a cleavable        linker, or a non-polymeric linker group. In some embodiments, X        is a bond. In some embodiments, X is a C₁-C₆ alkyl group. In        some embodiments, Y is a C₁-C₆ alkyl group. In some embodiments,        X is a homobifunctional linker or a heterobifunctional linker,        optionally conjugated to a C₁-C₆ alkyl group. In some        embodiments, Y is a homobifunctional linker or a        heterobifunctional linker. In some embodiments, the binding        moiety is an antibody or binding fragment thereof. In some        embodiments, the antibody or binding fragment thereof comprises        a humanized antibody or binding fragment thereof, chimeric        antibody or binding fragment thereof, monoclonal antibody or        binding fragment thereof, monovalent Fab′, divalent Fab2,        single-chain variable fragment (scFv), diabody, minibody,        nanobody, single-domain antibody (sdAb), or camelid antibody or        binding fragment thereof. In some embodiments, C is polyethylene        glycol. In some embodiments, C has a molecular weight of about        5000 Da. In some embodiments, A-X is conjugated to the 5′ end of        B and Y—C is conjugated to the 3′ end of B. In some embodiments,        Y—C is conjugated to the 5′ end of B and A-X is conjugated to        the 3′ end of B. In some embodiments, A-X, Y—C or a combination        thereof is conjugated to an internucleotide linkage group. In        some embodiments, methods further comprise D. In some        embodiments, D is conjugated to C or to A. In some embodiments,        D is conjugated to the molecule conjugate of Formula (II)        according to Formula (IV):        (A-X—B—Y—C_(c))L-D   Formula IV    -   wherein,    -   A comprises a binding moiety;    -   B consists of a polynucleotide;    -   C consists of a polymer;    -   X consists of a bond or first linker;    -   Y is a bond or second linker;    -   L consists of a bond or third linker;    -   D consists of an endosomolytic moiety; and    -   c is an integer between 0 and 1; and    -   wherein the polynucleotide comprises at least one 2′ modified        nucleotide, at least one modified internucleotide linkage, or an        inverted abasic moiety; and D is conjugated anywhere on A, B, or        C.        In some embodiments, D is INF7 or melittin. In some embodiments,        L is a C₁-C₆ alkyl group. In some embodiments, L is a        homobifunctional linker or a heterobifunctional linker. In some        embodiments, methods further comprise at least a second binding        moiety A. In some embodiments, the at least second binding        moiety A is conjugated to A, to B, or to C. In some embodiments,        the method is an in vivo method. In some embodiments, the method        is an in vitro method. In some embodiments, the subject is a        human.

Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising: a molecule obtained by any one of the methodsdisclosed herein and a pharmaceutically acceptable excipient. In someembodiments, the pharmaceutical composition is formulated as ananoparticle formulation. In some embodiments, the pharmaceuticalcomposition is formulated for parenteral, oral, intranasal, buccal,rectal, or transdermal administration.

Disclosed herein, in certain embodiments, are kits comprising a moleculeobtained by any one of the methods disclosed herein.

Disclosed herein, in certain embodiments, are compositions comprising apolynucleic acid molecule conjugate, wherein the polynucleic acidmolecule conjugate comprises a polynucleotide comprising a sequencehaving at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to SEQ ID NOs: 54-972. Disclosed herein,in certain embodiments, are compositions comprising a polynucleic acidmolecule conjugate, wherein the polynucleic acid molecule conjugatecomprises a polynucleotide comprising a sequence having at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNOs: 54-972. In certain embodiments, the polynucleic acid moleculeconjugate comprises a structure of Formula (I):A-X—B   Formula I

-   -   wherein,    -   A comprises a binding moiety;    -   B consists of the polynucleotide; and    -   X consists of a bond or first linker.        In certain embodiments, the polynucleic acid molecule conjugate        comprises a structure of Formula (II):        A-X—B—Y—C   Formula II    -   wherein,    -   A comprises a binding moiety;    -   B consists of the polynucleotide;    -   C consists of a polymer;    -   X consists of a bond or first linker; and    -   Y consists of a bond or second linker.        In certain embodiments, the polynucleic acid molecule conjugate        comprises a structure of Formula (III):        A-X—C—Y—B   Formula III    -   wherein,    -   A comprises a binding moiety;    -   B consists of the polynucleotide;    -   C consists of a polymer;    -   X consists of a bond or first linker; and    -   Y consists of a bond or second linker.        In certain embodiments, the at least one 2′ modified nucleotide        comprises a morpholino, 2′-O-methyl, 2′-O-methoxyethyl        (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro,        2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl        (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),        T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or        2′-O—N-methylacetamido (2′-O-NMA) modified nucleotide. In        certain embodiments, the at least one 2′ modified nucleotide        comprises a morpholino.

Disclosed herein, in certain embodiments, are methods of treating adisease or disorder comprising: administering to a subject a polynucleicacid molecule conjugate; wherein the polynucleic acid molecule conjugatecomprises a target cell binding moiety and a targeted pre-mRNA specificsplice modulating polynucleic acid moiety; wherein the target cellbinding moiety specifically binds to a targeted cell, and the targetedpre-mRNA specific splice modulating polynucleic acid moiety inducesinsertion, deletion, duplication, or alteration of a targeted pre-mRNAtranscript in the targeted cell to induce a splicing event in thetargeted pre-mRNA transcript to generate a mRNA transcript; and whereinthe mRNA transcript encodes a protein that is modified when compared tothe same protein in untreated target cells, thereby treating the diseaseor disorder in the subject. In certain embodiments, the splicing eventis exon skipping. In certain embodiments, the splicing event is exoninclusion. In certain embodiments, the disease or disorder is furthercharacterized by one or more mutations in the pre-mRNA. In certainembodiments, the disease or disorder comprises a neuromuscular disease,a genetic disease, cancer, a hereditary disease, or a cardiovasculardisease. In certain embodiments, the disease or disorder is musculardystrophy. In certain embodiments, the disease or disorder is Duchennemuscular dystrophy. In certain embodiments, the splicing event is ofexon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of DMD gene. Incertain embodiments, the splicing event is of exon 23 of DMD gene. Incertain embodiments, the splicing event is of an exon of PAH, MSTN, orK-Ras gene. In certain embodiments, the polynucleic acid moleculeconjugate comprises a structure of Formula (I):A-X—B   Formula I

-   -   wherein,    -   A comprises a binding moiety;    -   B consists of a polynucleotide; and    -   X consists of a bond or first linker.        In certain embodiments, the polynucleic acid molecule conjugate        comprises a structure of Formula (II):        A-X—B—Y—C   Formula II    -   wherein,    -   A comprises a binding moiety;    -   B consists of a polynucleotide;    -   C consists of a polymer;    -   X consists a bond or first linker; and    -   Y consists of a bond or second linker.        In certain embodiments, the polynucleic acid molecule conjugate        comprises a structure of Formula (III):        A-X—C—Y—B   Formula III    -   wherein,    -   A comprises a binding moiety;    -   B consists of a polynucleotide;    -   C consists of a polymer;    -   X consists of a bond or first linker; and    -   Y consists of a bond or second linker.        In certain embodiments, the polynucleic acid molecule conjugate        optionally comprises at least one 2′ modified nucleotide, at        least one modified internucleotide linkage, or at least one        inverted abasic moiety. In certain embodiments, the at least one        2′ modified nucleotide comprises a morpholino, 2′-O-methyl,        2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,        T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP),        2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl        (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or        2′-O—N-methylacetamido (2′-O-NMA) modified nucleotide. In        certain embodiments, the at least one 2′ modified nucleotide        comprises locked nucleic acid (LNA), ethylene nucleic acid        (ENA), or a peptide nucleic acid (PNA). In certain embodiments,        the at least one 2′ modified nucleotide comprises a morpholino.        In certain embodiments, the at least one inverted basic moiety        is at least one terminus. In certain embodiments, the at least        one modified internucleotide linkage comprises a        phosphorothioate linkage or a phosphorodithioate linkage. In        certain embodiments, the polynucleic acid molecule comprises at        least from about 10 to about 30 nucleotides in length. In        certain embodiments, the polynucleic acid molecule comprises at        least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or        99% modification. In certain embodiments, the polynucleic acid        molecule comprises a single strand. In certain embodiments, the        polynucleic acid molecule comprises two or more strands. In        certain embodiments, the polynucleic acid molecule comprises a        first polynucleotide and a second polynucleotide hybridized to        the first polynucleotide to form a double-stranded polynucleic        acid molecule. In certain embodiments, the second polynucleotide        comprises at least one modification. In certain embodiments, the        first polynucleotide and the second polynucleotide comprise RNA        molecules. In certain embodiments, the first polynucleotide and        the second polynucleotide comprise siRNA molecules. In certain        embodiments, X is a bond. In certain embodiments, X and Y are        independently a bond, a degradable linker, a non-degradable        linker, a cleavable linker, or a non-polymeric linker group. In        certain embodiments, X and Y are independently a bond, a        degradable linker, a non-degradable linker, a cleavable linker,        or a non-polymeric linker group. In certain embodiments, X is a        C₁-C₆ alkyl group. In certain embodiments, X or Y is a C₁-C₆        alkyl group. In certain embodiments, X or Y is a C₁-C₆ alkyl        group. In certain embodiments, the binding moiety is an antibody        or binding fragment thereof. In certain embodiments, the binding        moiety is an antibody or binding fragment thereof. In certain        embodiments, the binding moiety is an antibody or binding        fragment thereof. In certain embodiments, C is polyethylene        glycol. In certain embodiments, C is polyethylene glycol. In        certain embodiments, A-X is conjugated to the 5′ end of B and        Y—C is conjugated to the 3′ end of B. In certain embodiments,        Y—C is conjugated to the 5′ end of B and A-X is conjugated to        the 3′ end of B. In certain embodiments, methods further        comprise D. In certain embodiments, D is conjugated to C or        to A. In certain embodiments, methods further comprise at least        a second binding moiety A. In certain embodiments, methods        further comprise at least a second binding moiety A. In certain        embodiments, methods further comprise at least a second binding        moiety A.

Disclosed herein, in certain embodiments, are methods of inducing asplicing event in a targeted pre-mRNA transcript, comprising: (a)contacting a target cell with a polynucleic acid molecule conjugate,wherein the polynucleic acid molecule conjugate comprises a target cellbinding moiety and a targeted pre-mRNA splice modulating polynucleicacid moiety; (b) hybridizing the targeted pre-mRNA splice modulatingpolynucleic acid moiety to the targeted pre-mRNA transcript within thetarget cell to induce the splicing event in the targeted pre-mRNAtranscript to produce a mRNA transcript; and (c) optionally, translatingthe mRNA transcript of step (b) in the target cell to produce a protein.In certain embodiments, the splicing event is exon skipping. In certainembodiments, the splicing event is exon inclusion. In certainembodiments, the targeted pre-mRNA transcript induces a disease ordisorder. In certain embodiments, the disease or disorder comprises aneuromuscular disease, a genetic disease, cancer, a hereditary disease,or a cardiovascular disease. In certain embodiments, the polynucleicacid molecule conjugate:

-   -   a) comprises a structure of Formula (I):        A-X—B   Formula I    -   wherein,    -   A comprises a binding moiety;    -   B consists of the polynucleotide; and    -   X consists of a bond or first linker;    -   b) comprises a structure of Formula (II):        A-X—B—Y—C   Formula II    -   wherein,    -   A comprises a binding moiety;    -   B consists of the polynucleotide;    -   C consists of a polymer;    -   X consists of a bond or first linker; and    -   Y consists of a bond or second linker; or    -   c) comprises a structure of Formula (III):        A-X—C—Y—B   Formula III    -   wherein,    -   A comprises a binding moiety;    -   B consists of the polynucleotide;    -   C consists of a polymer;    -   X consists of a bond or first linker; and    -   Y consists of a bond or second linker.        In certain embodiments, the polynucleic acid molecule conjugate        optionally comprises at least one 2′ modified nucleotide, at        least one modified internucleotide linkage, or at least one        inverted abasic moiety. In certain embodiments, the at least one        2′ modified nucleotide comprises a morpholino, 2′-O-methyl,        2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,        T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP),        2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl        (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or        2′-O—N-methylacetamido (2′-O-NMA) modified nucleotide. In        certain embodiments, the at least one 2′ modified nucleotide        comprises locked nucleic acid (LNA), ethylene nucleic acid        (ENA), peptide nucleic acid (PNA). In certain embodiments, the        at least one 2′ modified nucleotide comprises a morpholino. In        certain embodiments, the at least one inverted basic moiety is        at least one terminus. In certain embodiments, the at least one        modified internucleotide linkage comprises a phosphorothioate        linkage or a phosphorodithioate linkage. In certain embodiments,        the polynucleic acid molecule comprises at least from about 10        to about 30 nucleotides in length. In certain embodiments, the        polynucleic acid molecule comprises at least about 15%, 20%,        30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modification. In        certain embodiments, the polynucleic acid molecule comprises at        least about 3, about 4, about 5, about 6, about 7, about 8,        about 9, about 10, about 11, about 12, about 13, about 14, about        15, about 16, about 17, about 18, about 19, about 20, about 21,        about 22 or more modifications. In certain embodiments, X and Y        are independently a bond, a degradable linker, a non-degradable        linker, a cleavable linker, or a non-polymeric linker group. In        certain embodiments, X is a bond. In certain embodiments, X is a        C₁-C₆ alkyl group. In certain embodiments, Y is a C₁-C₆ alkyl        group. In certain embodiments, X is a homobifunctional linker or        a heterobifunctional linker, optionally conjugated to a C₁-C₆        alkyl group. In certain embodiments, Y is a homobifunctional        linker or a heterobifunctional linker. In certain embodiments,        the binding moiety is an antibody or binding fragment thereof.        In certain embodiments, C is polyethylene glycol. In certain        embodiments, A-X is conjugated to the 5′ end of B and Y—C is        conjugated to the 3′ end of B. In certain embodiments, Y—C is        conjugated to the 5′ end of B and A-X is conjugated to the 3′        end of B. In certain embodiments, A-X, Y—C or a combination        thereof is conjugated to an internucleotide linkage group. In        certain embodiments, methods further comprise D. In certain        embodiments, D is conjugated to C or to A. In certain        embodiments, methods further comprise at least a second binding        moiety A.

Disclosed herein, in certain embodiments, are polynucleic acid moleculeconjugate compositions comprising a target cell binding moiety and atargeted pre-mRNA specific splice modulating polynucleic acid moietywherein the targeted pre-mRNA specific splice modulating polynucleicacid moiety comprises a sequence having at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQID NOs: 54-972. In certain embodiments, the polynucleic acid moleculeconjugate:

-   -   a) comprises a structure of Formula (I):        A-X—B   Formula I    -   wherein,    -   A comprises a binding moiety;    -   B consists of the polynucleotide; and    -   X consists of a bond or first linker;    -   b) comprises a structure of Formula (II):        A-X—B—Y—C   Formula II    -   wherein,    -   A comprises a binding moiety;    -   B consists of the polynucleotide;    -   C consists of a polymer;    -   X consists of a bond or first linker; and    -   Y consists of a bond or second linker; or    -   c) comprises a structure of Formula (III):        A-X—C—Y—B   Formula III    -   wherein,    -   A comprises a binding moiety;    -   B consists of the polynucleotide;    -   C consists of a polymer;    -   X consists of a bond or first linker; and    -   Y consists of a bond or second linker.        In certain embodiments, the pharmaceutical composition is        formulated as a nanoparticle formulation.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a phosphorodiamidate morpholino oligomer (PMO) sequencewith end nucleotides expanded (SEQ ID NO: 28).

FIG. 2A depicts a phosphorothioate antisense oligonucleotide (PS ASO)sequence with end nucleotides expanded (SEQ ID NO: 29).

FIG. 2B depicts a fully expanded phosphorothioate antisenseoligonucleotide (PS ASO) sequence (SEQ ID NO: 29).

FIG. 3 depicts methods used to quantify skipped DMD mRNA in total RNAusing Taqman qPCR.

FIG. 4 depicts a chromatogram of anti-CD71 mAb-PMO reaction mixtureproduced with hydrophobic interaction chromatography (HIC) method 2.

FIG. 5A depicts a chromatogram of anti-CD71 mAb produced using sizeexclusion chromatography (SEC) method 1.

FIG. 5B depicts a chromatogram of anti-CD71 mAb-PMO DAR 1,2 producedusing size exclusion chromatography (SEC) method 1.

FIG. 5C depicts a chromatogram of anti-CD71 mAb-PMO DAR >2 producedusing size exclusion chromatography (SEC) method 1.

FIG. 6A depicts a chromatogram of anti-CD71 mAb produced usinghydrophobic interaction chromatography (HIC) method 2.

FIG. 6B depicts a chromatogram of purified anti-CD71 mAb-PMO DAR 1,2conjugate produced using hydrophobic interaction chromatography (HIC)method 2.

FIG. 6C depicts a chromatogram of purified anti-CD71 mAb-PMO DAR >2conjugate produced using hydrophobic interaction chromatography (HIC)method 2.

FIG. 7A depicts a chromatogram of fast protein liquid chromatography(FPLC) purification of anti-CD71 Fab-PMO using hydrophobic interactionchromatography (HIC) method 3.

FIG. 7B depicts a chromatogram of anti-CD71 Fab produced using SECmethod 1.

FIG. 7C depicts a chromatogram of anti-CD71 Fab-PMO DAR 1 conjugateproduced using SEC method 1.

FIG. 7D depicts a chromatogram of anti-CD71 Fab-PMO DAR 2 conjugateproduced using SEC method 1.

FIG. 7E depicts a chromatogram of anti-CD71 Fab-PMO DAR 3 conjugateproduced using SEC method 1.

FIG. 7F depicts a chromatogram of anti-CD71 Fab produced using HICmethod 4.

FIG. 7G depicts a chromatogram of anti-CD71 Fab-PMO DAR 1 conjugateproduced using HIC method 4.

FIG. 7H depicts a chromatogram of anti-CD71 Fab-PMO DAR 2 conjugateproduced using HIC method 4.

FIG. 7I depicts a chromatogram of anti-CD71 Fab-PMO DAR 3 conjugateproduced using HIC method 4.

FIG. 8A depicts a chromatogram of anti-CD71 mAb-PS ASO reaction mixtureproduced with SAX method 2.

FIG. 8B depicts a chromatogram of anti-CD71 mAb produced using SECmethod 1.

FIG. 8C depicts a chromatogram of anti-CD71 mAb-PS ASO DAR 1 conjugateproduced using SEC method 1.

FIG. 8D depicts a chromatogram of anti-CD71 mAb-PS ASO DAR 2 conjugateproduced using SEC method 1.

FIG. 8E depicts a chromatogram of anti-CD71 mAb-PS ASO DAR 3 conjugateproduced using SEC method 1.

FIG. 8F depicts a chromatogram of anti-CD71 mAb-PS ASO DAR 1 conjugateproduced using SAX method 2.

FIG. 8G depicts a chromatogram of anti-CD71 mAb-PS ASO DAR 2 conjugateproduced using SAX method 2.

FIG. 8H depicts a chromatogram of anti-CD71 mAb-PS ASO DAR 3 conjugateproduced using SAX method 2.

FIG. 9 depicts an agarose gel from nested PCR detecting exon 23 skippingin differentiated C2C12 cells using PMO and anti-CD71 mAb-PMO conjugate.

FIG. 10 depicts an agarose gel from nested PCR detecting exon 23skipping in differentiated C2C12 cells using PMO, anti-CD71 mAb-PMO, andanti-CD71 Fab-PMO conjugates.

FIG. 11 depicts an agarose gel from nested PCR detecting exon 23skipping in differentiated C2C12 cells PMO, ASO, conjugated anti-CD71mAb-ASO of DAR1 (“ASC-DAR1”), conjugated anti-CD71 mAb-ASO of DAR2(“ASC-DAR2”), and conjugated anti-CD71 mAb-ASO of DAR3 (“ASC-DAR3”).

FIG. 12A depicts an agarose gel from nested PCR detecting exon 23skipping in gastrocnemius muscle of wild-type mice administered a singleintravenous injection of anti-CD71 mAb-PMO conjugate.

FIG. 12B is a graph of quantification of PCR products from gastrocnemiusmuscle.

FIG. 12C is a graph of quantification of in vivo exon skipping usingTaqman qPCR from gastrocnemius muscle from wild-type mice.

FIG. 13A depicts an agarose gel from nested PCR detecting exon 23skipping in heart muscle from wild-type mice after a single intravenousinjection.

FIG. 13B is a graph of quantification of PCR products from heart muscle.

FIG. 14 depicts sequencing data of DNA fragments from skipped andwild-type PCR products (SEQ ID NOS 976-977, respectively).

FIG. 15A is a graph of quantification of in vivo exon skipping in wildtype mice in gastrocnemius muscle using Taqman qPCR.

FIG. 15B is a graph of quantification of in vivo exon skipping in wildtype mice in gastrocnemius muscle using nested PCR.

FIG. 15C is a graph of quantification of in vivo exon skipping in wildtype mice in diaphragm muscle using Taqman qPCR.

FIG. 15D is a graph of quantification of in vivo exon skipping in wildtype mice in diaphragm muscle using nested PCR.

FIG. 15E is a graph of quantification of in vivo exon skipping in wildtype mice in heart muscle using Taqman qPCR.

FIG. 15F is a graph of quantification of in vivo exon skipping in wildtype mice in heart muscle using nested PCR.

FIG. 16A depicts an agarose gel from PCR detecting CD71 mAb-PMOconjugate induction of MSTN exon 2 skipping in diaphragm muscle tissuesin wild type mice after a single intravenous (i.v.) injection.

FIG. 16B depicts an agarose gel from PCR detecting CD71 mAb-PMOconjugate induction of MSTN exon 2 skipping in heart muscle tissues inwild type mice after a single intravenous (i.v.) injection.

FIG. 16C depicts an agarose gel from PCR detecting CD71 mAb-PMOconjugate induction of MSTN exon 2 skipping in gastrocnemius muscletissues in wild type mice after a single intravenous (i.v.) injection.

FIG. 17 depicts an agarose gel from PCR detecting ASGPR mAb-PMOconjugate induction of PAH exon 11 skipping in primary mousehepatocytes.

FIG. 18 depicts an agarose gel from PCR detecting ASGPR mAb-PMOconjugate induction of PAH exon 11 skipping in livers from wild typemice after a single intravenous (i.v.) injection.

FIG. 19A-FIG. 19L illustrate cartoon representations of moleculesdescribed herein.

DETAILED DESCRIPTION OF THE DISCLOSURE

Nucleic acid (e.g., RNAi) therapy is a targeted therapy with highselectivity and specificity. However, in some instances, nucleic acidtherapy is also hindered by poor intracellular uptake, insufficientintracellular concentrations in target cells, and low efficacy. Toaddress these issues, various modifications of the nucleic acidcomposition are explored, such as for example, novel linkers for betterstabilizing and/or lower toxicity, optimization of binding moiety forincreased target specificity and/or target delivery, and nucleic acidpolymer modifications for increased stability and/or reduced off-targeteffect.

In some instances, one such area where oligonucleotide is used is fortreating muscular dystrophy. Muscular dystrophy encompasses severaldiseases that affect the muscle. Duchenne muscular dystrophy is a severeform of muscular dystrophy and caused by mutations in the DMD gene. Insome instances, mutations in the DMD gene disrupt the translationalreading frame and results in non-functional dystrophin protein.

Described herein, in certain embodiments, are methods and compositionsrelating nucleic acid therapy to induce an insertion, deletion,duplication, or alteration in an incorrectly spliced mRNA transcript toinduce exon skipping or exon inclusion, which is used to restore thetranslational reading frame. In some embodiments, also described hereininclude methods and compositions for treating a disease or disordercharacterized by an incorrectly processed mRNA transcript, in whichafter removal of an exon, the mRNA is capable of encoding a functionalprotein, thereby treating the disease or disorder. In additionalembodiments, described herein include pharmaceutical compositions andkits for treating the same.

RNA Processing

RNA has a central role in regulation of gene expression and cellphysiology. Proper processing of RNA is important for translational offunctional protein. Alterations in RNA processing such as a result ofincorrect splicing of RNA can result in disease. For example, mutationsin a splice site causes exposure of a premature stop codon, a loss of anexon, or inclusion of an intron. In some instances, alterations in RNAprocessing results in an insertion, deletion, or duplication. In someinstances, alterations in RNA processing results in an insertion,deletion, or duplication of an exon. Alterations in RNA processing, insome cases, results in an insertion, deletion, or duplication of anintron.

Alternative transcriptional or splicing events include, but are notlimited to, exon skipping, alternative 3′ splice site selection,alternative 5′ splice site selection, intron retention, mutuallyexclusive exons, alternative promoter usage, and alternativepolyadenylation. Splicing events, in some embodiments, results in aninsertion, deletion, or duplication of an exon, for example, by exonskipping or exon inclusion.

Exon Skipping

Exon skipping is a form of RNA splicing. In some cases, exon skippingoccurs when an exon is skipped over or is spliced out of the processedmRNA. As a result of exon skipping, the processed mRNA does not containthe skipped exon. In some instances, exon skipping results in expressionof an altered product.

In some instances, antisense oligonucleotides (AONs) are used to induceexon skipping. In some instances, AONs are short nucleic acid sequencesthat bind to specific mRNA or pre-mRNA sequences. For example, AONs bindsplice sites or exonic enhancers. In some instances, binding of AONs tospecific mRNA or pre-mRNA sequences generates double-stranded regions.In some instances, formation of double-stranded regions occurs at siteswhere the spliceosome or proteins associated with the spliceosome wouldnormally bind and causes exons to be skipped. In some instances,skipping of exons results in restoration of the transcript reading frameand allows for production of a partially functional protein.

Exon Inclusion

In some instances, a mutation in RNA results in exon skipping. In somecases, a mutation is at least one of at the splice site, near the splicesite, and at a distance from the splice site. In some instances, themutations result in at least one of inactivating or weakening the splicesite, disrupting exon splice enhancer or intron splice enhancer, andcreating an exon splice silencer or intron splice enhancer. Mutations insome instances alter RNA secondary structure. In some cases, a mutationalters a RNA secondary structure result in disrupting the accessibilityof signals important for exon recognition.

In some instances, use of AONs results in inclusion of the skipped exon.In some instances, the AONs bind to at least one of a splice site, asite near a splice site, and a site distant to a splice site. In somecases, AONs bind at site in the RNA to prevent disruption of an exonsplice enhancer or intron splice enhancer. In some instances, AONs bindat site in the RNA to prevent creation of an exon splice silencer orintron splice silencer.

Intron Retention

In some instances, a mutation in RNA results in intron retention. Intronretention results in an intron remaining in the mature mRNA transcript.In some instances, presence of a retained intron prevents or reducestranslation of a functional protein. In some instances, intron retentionoccurs in a coding region, a non-coding region, at the 5′ UTR, or at the3′ UTR. Where intron retention occurs in a coding region, in someinstances, the retained intron encodes amino acids in frame, or is inmisalignment which generates truncated proteins or non-functionalproteins due to stop codon or frame shifts. In some instances, theintron is retained between two exons, located at the 5′ UTR, or locatedat the 3′ UTR.

In some instances, AONs are used to hybridize to a partially processedmRNA to initiate removal of a retained intron. In some instances, theAONs hybridize to an intronic splicing enhancer or an intronic splicingsilencer. In some instances, the AONs hybridize at or a distance from a5′ splice site, 3′ splice site, branchpoint, polypyrimidine tract, anintron silencer site, a cryptic intron splice site, a pseudo splicesite, or an intron enhancer of the intron. In some instances, the AONshybridize to an internal region of the intron.

Indications

In some embodiments, a polynucleic acid molecule or a pharmaceuticalcomposition described herein is used for the treatment of a disease ordisorder characterized with a defective mRNA. In some embodiments, apolynucleic acid molecule or a pharmaceutical composition describedherein is used for the treatment of disease or disorder by inducing aninsertion, deletion, duplication, or alteration in an incorrectlyspliced mRNA transcript to induce a splicing event. In some embodiments,the splicing event is exon skipping or exon inclusion. In someembodiments, the splicing event is intron retention.

In some embodiments, a polynucleic acid molecule or a pharmaceuticalcomposition described herein is used for the treatment of disease ordisorder by inducing an insertion, deletion, duplication, or alterationin an incorrectly spliced mRNA transcript to induce exon skipping orexon inclusion.

A large percentage of human protein-coding genes are alternativelyspliced. In some instances, a mutation results in improperly spliced orpartially spliced mRNA. For example, a mutation is in at least one of asplice site in a protein coding gene, a silencer or enhancer sequence,exonic sequences, or intronic sequences. In some instances, a mutationresults in gene dysfunction. In some instances, a mutation results in adisease or disorder.

In some instances, a disease or disorder resulting from improperlyspliced or partially spliced mRNA includes, but not limited to, aneuromuscular disease, a genetic disease, cancer, a hereditary disease,or a cardiovascular disease.

In some instances, genetic diseases or disorders include an autosomaldominant disorder, an autosomal recessive disorder, X-linked dominantdisorder, X-linked recessive disorder, Y-linked disorder, mitochondrialdisease, or multifactorial or polygenic disorder.

In some instances, cardiovascular disease such as hypercholesterolemiaresults from improperly spliced or partially spliced mRNA. Inhypercholesterolemia, it has been shown that a single nucleotidepolymorphism in exon 12 of the low density lipoprotein receptor (LDLR)promotes exon skipping.

In some instances, improperly spliced or partially spliced mRNA resultsin cancer. For example, improperly spliced or partially spliced mRNAaffects cellular processes involved in cancer including, but not limitedto, proliferation, motility, and drug response. In some instances is asolid cancer or a hematologic cancer. In some instances, the cancer isbladder cancer, lung cancer, brain cancer, melanoma, breast cancer,Non-Hodgkin lymphoma, cervical cancer, ovarian cancer, colorectalcancer, pancreatic cancer, esophageal cancer, prostate cancer, kidneycancer, skin cancer, leukemia, thyroid cancer, liver cancer, or uterinecancer.

Improperly spliced or partially spliced mRNA in some instances causes aneuromuscular disease or disorder. Exemplary neuromuscular diseasesinclude muscular dystrophy such as Duchenne muscular dystrophy, Beckermuscular dystrophy, facioscapulohumeral muscular dystrophy, congenitalmuscular dystrophy, or myotonic dystrophy. In some instances, musculardystrophy is genetic. In some instances, muscular dystrophy is caused bya spontaneous mutation. Becker muscular dystrophy and Duchenne musculardystrophy have been shown to involve mutations in the DMD gene, whichencodes the protein dystrophin. Facioscapulohumeral muscular dystrophyhas been shown to involve mutations in double homeobox, 4 (DUX4) gene.

In some instances, improperly spliced or partially spliced mRNA causesDuchenne muscular dystrophy. Duchenne muscular dystrophy results insevere muscle weakness and is caused by mutations in the DMD gene thatabolishes the production of functional dystrophin. In some instances,Duchenne muscular dystrophy is a result of a mutation in an exon in theDMD gene. In some instances, Duchenne muscular dystrophy is a result ofa mutation in at least one of exon 1, 2, 3, 4, 5, 6, 7, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78 and 79 in the DMD gene. In some instances, Duchenne musculardystrophy is a result of a mutation in at least one of exon 3, 4, 5, 6,7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 in the DMD gene. In someinstances, Duchenne muscular dystrophy is a result of a mutation in atleast one of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, and 55 in theDMD gene. In some instances, multiple exons are mutated. For example,mutation of exons 48-50 is common in Duchenne muscular dystrophypatients. In some instances, Duchenne muscular dystrophy is a result ofmutation of exon 51. In some instances, Duchenne muscular dystrophy is aresult of mutation of exon 23. In some instances, a mutation involves adeletion of an exon. In some instances, a mutation involves aduplication of an exon. In some instances, a mutation involves a pointmutation in an exon. For example, it has been shown that some patientshave a nonsense point mutation in exon 51 of the DMD gene.

In some instances, a polynucleic acid molecule or a pharmaceuticalcomposition described herein is used for the treatment of musculardystrophy. In some instances, a polynucleic acid molecule or apharmaceutical composition described herein is used for the treatment ofDuchenne muscular dystrophy, Becker muscular dystrophy,facioscapulohumeral muscular dystrophy, congenital muscular dystrophy,or myotonic dystrophy. In some instances, a polynucleic acid molecule ora pharmaceutical composition described herein is used for the treatmentof Duchenne muscular dystrophy.

Polynucleic Acid Molecule

In some embodiments, a polynucleic acid molecule described herein thatinduces an insertion, deletion, duplication, or alteration in anincorrectly spliced mRNA transcript to induce exon skipping or exoninclusion. In some instances, the polynucleic acid molecule restores thetranslational reading frame. In some instances, the polynucleic acidmolecule results in a functional and truncated protein.

In some instances, a polynucleic acid molecule targets a mRNA sequence.In some instances, the polynucleic acid molecule targets a splice site.In some instances, the polynucleic acid molecule targets acis-regulatory element. In some instances, the polynucleic moleculetargets a trans-regulatory element. In some instances, the polynucleicacid molecule targets exonic splice enhancers or intronic spliceenhancers. In some instances, the polynucleic acid molecule targetsexonic splice silencers or intronic splice silencers.

In some instances, a polynucleic acid molecule targets a sequence foundin introns or exons. For example, the polynucleic acid molecule targetsa sequence found in an exon that mediates splicing of said exon. In someinstances, the polynucleic acid molecule targets an exon recognitionsequence. In some instances, the polynucleic acid molecule targets asequence upstream of an exon. In some instances, the polynucleic acidmolecule targets a sequence downstream of an exon.

As described above, a polynucleic acid molecule targets an incorrectlyprocessed mRNA transcript which results in a disease or disorder notlimited to a neuromuscular disease, a genetic disease, cancer, ahereditary disease, or a cardiovascular disease.

In some instances, a polynucleic acid molecule targets an exon that ismutated in a gene that causes a disease or disorder. Exemplary diseasesor disorders include, but are not limited to, familial dysautonomia(FD), spinal muscular atrophy (SMA), medium-chain acyl-CoA dehydrogenase(MCAD) deficiency, Hutchinson-Gilford progeria syndrome (HGPS), myotonicdystrophy type I (DM1), myotonic dystrophy type II (DM2), autosomaldominant retinitis pigmentosa (RP), Duchenne muscular dystrophy (DMD),microcephalic steodysplastic primordial dwarfism type 1 (MOPD1)(Taybi-Linder syndrome (TALS)), frontotemporal dementia withparkinsonism-17 (FTDP-17), Fukuyama congenital muscular dystrophy(FCMD), amyotrophic lateral sclerosis (ALS), hypercholesterolemia, andcystic fibrosis (CF). Exemplary genes that are involved in the diseaseor disorder include, but are not limited to, IKBKAP, SMN2, MCAD, LMNA,DMPK, ZNF9, MAPT, FKTN, TDP-43, LDLR, CFTR, DMD, PAH, MSTN, and K-Ras.In some embodiments, the gene is DMD, PAH, MSTN, or K-Ras.

In some instances, a polynucleic acid molecule described herein targetsa region that is at the exon-intron junction of an exon of a gene thatcauses a disease or disorder. In some embodiments, the gene is IKBKAP,SMN2, MCAD, LMNA, DMPK, ZNF9, MAPT, FKTN, TDP-43, LDLR, CFTR, DMD, PAH,MSTN, or K-Ras. In some embodiments, a polynucleic acid moleculedescribed herein targets a region that is at the exon-intron junction ofexon 1, 2, or 3 of MSTN. In some embodiments, a polynucleic acidmolecule described herein targets a region that is at the exon-intronjunction of exon 2 of MSTN. In some embodiments, a polynucleic acidmolecule described herein targets a region that is at the exon-intronjunction of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, or 21 of PAH. In some embodiments, a polynucleic acidmolecule described herein targets a region that is at the exon-intronjunction of exon 11 of PAH.

In some instances, the polynucleic acid molecule hybridizes to a targetregion that is at either the 5′ intron-exon junction or the 3′exon-intron junction of at least one of an exon of a gene that causes adisease or disorder. In some embodiments, the gene is IKBKAP, SMN2,MCAD, LMNA, DMPK, ZNF9, MAPT, FKTN, TDP-43, LDLR, CFTR, DMD, PAH, MSTN,or K-Ras. In some embodiments, a polynucleic acid molecule describedherein targets either the 5′ intron-exon junction or the 3′ exon-intronjunction of exon 1, 2, or 3 of MSTN. In some embodiments, a polynucleicacid molecule described herein targets a region that is either the 5′intron-exon junction or the 3′ exon-intron junction of exon 2 of MSTN.In some embodiments, a polynucleic acid molecule described hereintargets a region that is either the 5′ intron-exon junction or the 3′exon-intron junction of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or 21 of PAH. In some embodiments, apolynucleic acid molecule described herein targets a region that iseither the 5′ intron-exon junction or the 3′ exon-intron junction ofexon 11 of PAH.

In some cases, the polynucleic acid molecule hybridizes to a targetregion that is at the 5′ intron-exon junction of at least one of exon ofa gene that causes a disease or disorder. In some embodiments, the geneis IKBKAP, SMN2, MCAD, LMNA, DMPK, ZNF9, MAPT, FKTN, TDP-43, LDLR, CFTR,DMD, PAH, MSTN, or K-Ras. In some embodiments, a polynucleic acidmolecule described herein targets a region that is at the 5′ intron-exonjunction of exon 1, 2, or 3 of MSTN. In some embodiments, a polynucleicacid molecule described herein targets a region that is at the 5′intron-exon junction of exon 2 of MSTN. In some embodiments, apolynucleic acid molecule described herein targets a region that is atthe 5′ intron-exon junction of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 of PAH. In some embodiments, apolynucleic acid molecule described herein targets a region that is atthe 5′ intron-exon junction of exon 11 of PAH.

In some cases, the polynucleic acid molecule hybridizes to a targetregion that is at the 3′ exon-intron junction of at least one of exon ofa gene that causes a disease or disorder. In some embodiments, the geneis IKBKAP, SMN2, MCAD, LMNA, DMPK, ZNF9, MAPT, FKTN, TDP-43, LDLR, CFTR,DMD, PAH, MSTN, or K-Ras. In some embodiments, a polynucleic acidmolecule described herein targets a region that is at the 3′ exon-intronjunction of exon 1, 2, or 3 of MSTN. In some embodiments, a polynucleicacid molecule described herein targets a region that is at the 3′exon-intron junction of exon 2 of MSTN. In some embodiments, apolynucleic acid molecule described herein targets a region that is atthe 3′ exon-intron junction of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 of PAH. In some embodiments, apolynucleic acid molecule described herein targets a region that is atthe 3′ exon-intron junction of exon 11 of PAH.

In some cases, the polynucleic acid molecule described herein targets asplice site of an exon of a gene that causes a disease or disorder. Insome embodiments, the gene is IKBKAP, SMN2, MCAD, LMNA, DMPK, ZNF9,MAPT, FKTN, TDP-43, LDLR, CFTR, DMD, PAH, MSTN, or K-Ras. In someembodiments, a polynucleic acid molecule described herein targets asplice site of exon 1, 2, or 3 of MSTN. In some embodiments, apolynucleic acid molecule described herein targets a splice site of exon2 of MSTN. In some embodiments, a polynucleic acid molecule describedherein targets a splice site of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 of PAH. In some embodiments, apolynucleic acid molecule described herein targets a splice site of exon11 of PAH. As used herein, a splice site includes a canonical splicesite, a cryptic splice site or an alternative splice site that iscapable of inducing an insertion, deletion, duplication, or alterationin an incorrectly spliced mRNA transcript to induce exon skipping orexon inclusion.

In some instances, a polynucleic acid molecule described herein targetsa region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt,100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 ntupstream (or from the 5′) of an exon of a gene that causes a disease ordisorder. In some embodiments, the gene is IKBKAP, SMN2, MCAD, LMNA,DMPK, ZNF9, MAPT, FKTN, TDP-43, LDLR, CFTR, DMD, PAH, MSTN, or K-Ras. Insome instances, a polynucleic acid molecule described herein targets aregion at least 1000 nt, 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt,60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt upstream (or from the5′) of exon 1, 2, or 3 of the MSTN gene. In some instances, apolynucleic acid molecule described herein targets a region at least1000 nt, 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40nt, 30 nt, 20 nt, 10 nt, or 5 nt upstream (or from the 5′) of exon 2 ofthe MSTN gene. In some instances, a polynucleic acid molecule describedherein targets a region at least 1000 nt, 500 nt, 400 nt, 300 nt, 200nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 ntupstream (or from the 5′) of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or 21 of PAH gene. In some instances, apolynucleic acid molecule described herein targets a region at least1000 nt, 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40nt, 30 nt, 20 nt, 10 nt, or 5 nt upstream (or from the 5′) of exon 11 ofthe PAH gene.

In some instances, the polynucleic acid molecule hybridizes to a targetregion that is upstream (or 5′) to at least one of an exon of a genethat causes a disease or disorder. In some embodiments, the gene isIKBKAP, SMN2, MCAD, LMNA, DMPK, ZNF9, MAPT, FKTN, TDP-43, LDLR, CFTR,DMD, PAH, MSTN, or K-Ras. In some instances, the polynucleic acidmolecule hybridizes to a target region that is upstream (or 5′) to atleast one of exon 1, 2, or 3 of the MSTN gene. In some instances, thepolynucleic acid molecule hybridizes to a target region that is about 5,10, 15, 20, 50, 100, 200, 300, 400 or 500 bp upstream (or 5′) to atleast one of exon 2 of the MSTN gene. In some instances, the polynucleicacid molecule hybridizes to a target region that is upstream (or 5′) toat least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or 21 of the PAH gene. In some instances, thepolynucleic acid molecule hybridizes to a target region that is about 5,10, 15, 20, 50, 100, 200, 300, 400 or 500 bp upstream (or 5′) to atleast one of exon 11 of the PAH gene.

In some instances, a polynucleic acid molecule described herein targetsa region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt,100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 ntdownstream (or from the 3′) of an exon of a gene that causes a diseaseor disorder. In some embodiments, the gene is IKBKAP, SMN2, MCAD, LMNA,DMPK, ZNF9, MAPT, FKTN, TDP-43, LDLR, CFTR, DMD, PAH, MSTN, or K-Ras. Insome instances, a polynucleic acid molecule described herein targets aregion at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt,100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 ntdownstream (or from the 3′) of exon 1, 2, or 3 of the MSTN gene. In someinstances, a polynucleic acid molecule described herein targets a regionat least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt,80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (orfrom the 3′) of exon 2 of the MSTN gene. In some instances, apolynucleic acid molecule described herein targets a region at least1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or from the3′) of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, or 21 of the PAH gene. In some instances, a polynucleic acidmolecule described herein targets a region at least 1000 nucleotides(nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or from the 3′) of exon 11of the PAH gene.

In some instances, the polynucleic acid molecule hybridizes to a targetregion that is downstream (or 3′) to at least one of an exon of a genethat causes a disease or disorder. In some embodiments, the gene isIKBKAP, SMN2, MCAD, LMNA, DMPK, ZNF9, MAPT, FKTN, TDP-43, LDLR, CFTR,DMD, PAH, MSTN, or K-Ras. In some instances, the polynucleic acidmolecule hybridizes to a target region that is about 5, 10, 15, 20, 50,100, 200, 300, 400 or 500 bp downstream (or 3′) to at least one of exon1, 2, or 3 of the MSTN gene. In some instances, the polynucleic acidmolecule hybridizes to a target region that is about 5, 10, 15, 20, 50,100, 200, 300, 400 or 500 bp downstream (or 3′) to at least one of exon2 of the MSTN gene. In some instances, the polynucleic acid moleculehybridizes to a target region that is about 5, 10, 15, 20, 50, 100, 200,300, 400 or 500 bp downstream (or 3′) to at least one of exon 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 ofthe PAH gene. In some instances, the polynucleic acid moleculehybridizes to a target region that is about 5, 10, 15, 20, 50, 100, 200,300, 400 or 500 bp downstream (or 3′) to at least one of exon 11 of thePAH gene.

In some instances, a polynucleic acid molecule described herein targetsan internal region within an exon of a gene that causes a disease ordisorder. In some embodiments, the gene is IKBKAP, SMN2, MCAD, LMNA,DMPK, ZNF9, MAPT, FKTN, TDP-43, LDLR, CFTR, DMD, PAH, MSTN, or K-Ras. Insome instances, a polynucleic acid molecule described herein targets aninternal region within exon 1, 2, or 3 of the MSTN gene. In someinstances, a polynucleic acid molecule described herein targets aninternal region within exon 2 of the MSTN gene. In some instances, apolynucleic acid molecule described herein targets an internal regionwithin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or 21 of the PAH gene. In some instances, a polynucleic acidmolecule described herein targets an internal region within exon 11 ofthe PAH gene.

In some cases, a polynucleic acid molecule targets an incorrectlyprocessed mRNA transcript which results in a neuromuscular disease ordisorder. In some cases, a neuromuscular disease or disorder is Duchennemuscular dystrophy, Becker muscular dystrophy, facioscapulohumeralmuscular dystrophy, congenital muscular dystrophy, or myotonicdystrophy. In some cases, a polynucleic acid molecule targets anincorrectly processed mRNA transcript which results in Duchenne musculardystrophy, Becker muscular dystrophy, facioscapulohumeral musculardystrophy, congenital muscular dystrophy, or myotonic dystrophy. In somecases, a polynucleic acid molecule targets an incorrectly processed mRNAtranscript which results in Duchenne muscular dystrophy.

In some instances, a polynucleic acid molecule targets an exon that ismutated in the DMD gene that causes Duchenne muscular dystrophy.Exemplary exons that are mutated in the DMD gene that causes Duchennemuscular dystrophy include, but not limited to, exon 3, 4, 5, 6, 7, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, and 63. In some instances, thepolynucleic acid molecule targets a sequence adjacent to a mutated exon.For example, if there is a deletion of exon 50, the polynucleic acidmolecule targets a sequence in exon 51 so that exon 51 is skipped. Inanother instance, if there is a mutation in exon 23, the polynucleicacid molecule targets a sequence in exon 22 so that exon 23 is skipped.

In some instances, a polynucleic acid molecule described herein targetsa region that is at the exon-intron junction of exon 3, 4, 5, 6, 7, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, or 63 of the DMD gene. In someinstances, a polynucleic acid molecule described herein targets a regionthat is at the exon-intron junction of exon 8, 23, 35, 43, 44, 45, 50,51, 52, 53, or 55 of the DMD gene. In some cases, a polynucleic acidmolecule described herein targets a region that is at the exon-intronjunction of exon 8 of the DMD gene. In some cases, a polynucleic acidmolecule described herein targets a region that is at the exon-intronjunction of exon 23 of the DMD gene. In some cases, a polynucleic acidmolecule described herein targets a region that is at the exon-intronjunction of exon 35 of the DMD gene. In some cases, a polynucleic acidmolecule described herein targets a region that is at the exon-intronjunction of exon 43 of the DMD gene. In some cases, a polynucleic acidmolecule described herein targets a region that is at the exon-intronjunction of exon 44 of the DMD gene. In some cases, a polynucleic acidmolecule described herein targets a region that is at the exon-intronjunction of exon 45 of the DMD gene. In some cases, a polynucleic acidmolecule described herein targets a region that is at the exon-intronjunction of exon 48 of the DMD gene. In some cases, a polynucleic acidmolecule described herein targets a region that is at the exon-intronjunction of exon 49 of the DMD gene. In some cases, a polynucleic acidmolecule described herein targets a region that is at the exon-intronjunction of exon 50 of the DMD gene. In some cases, a polynucleic acidmolecule described herein targets a region that is at the exon-intronjunction of exon 51 of the DMD gene. In some cases, a polynucleic acidmolecule described herein targets a region that is at the exon-intronjunction of exon 52 of the DMD gene. In some cases, a polynucleic acidmolecule described herein targets a region that is at the exon-intronjunction of exon 53 of the DMD gene. In some cases, a polynucleic acidmolecule described herein targets a region that is at the exon-intronjunction of exon 55 of the DMD gene.

In some instances, the polynucleic acid molecule hybridizes to a targetregion that is at either the 5′ intron-exon junction or the 3′exon-intron junction of at least one of exon 3, 4, 5, 6, 7, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, and 63 of the DMD gene. In some instances, thepolynucleic acid molecule hybridizes to a target region that is ateither the 5′ intron-exon junction or the 3′ exon-intron junction ofexon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene.

In some cases, the polynucleic acid molecule hybridizes to a targetregion that is at the 5′ intron-exon junction of at least one of exon 3,4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene. Insome cases, the polynucleic acid molecule hybridizes to a target regionthat is at the 5′ intron-exon junction of exon 8, 23, 35, 43, 44, 45,50, 51, 52, 53, or 55 of the DMD gene. In some cases, the polynucleicacid molecule hybridizes to a target region that is at the 5′intron-exon junction of exon 8 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the5′ intron-exon junction of exon 23 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the5′ intron-exon junction of exon 35 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the5′ intron-exon junction of exon 43 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the5′ intron-exon junction of exon 44 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the5′ intron-exon junction of exon 45 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the5′ intron-exon junction of exon 50 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the5′ intron-exon junction of exon 51 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the5′ intron-exon junction of exon 52 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the5′ intron-exon junction of exon 53 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the5′ intron-exon junction of exon 55 of the DMD gene.

In some cases, the polynucleic acid molecule hybridizes to a targetregion that is at the 3′ exon-intron junction of at least one of exon 3,4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene. Insome cases, the polynucleic acid molecule hybridizes to a target regionthat is at the 3′ exon-intron junction of exon 8, 23, 35, 43, 44, 45,50, 51, 52, 53, or 55 of the DMD gene. In some cases, the polynucleicacid molecule hybridizes to a target region that is at the 3′exon-intron junction of exon 8 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the3′ exon-intron junction of exon 23 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the3′ exon-intron junction of exon 35 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the3′ exon-intron junction of exon 43 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the3′ exon-intron junction of exon 44 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the3′ exon-intron junction of exon 45 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the3′ exon-intron junction of exon 50 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the3′ exon-intron junction of exon 51 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the3′ exon-intron junction of exon 52 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the3′ exon-intron junction of exon 53 of the DMD gene. In some cases, thepolynucleic acid molecule hybridizes to a target region that is at the3′ exon-intron junction of exon 55 of the DMD gene.

In some instances, a polynucleic acid molecule described herein targetsa splice site of exon 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,or 63 of the DMD gene. In some instances, a polynucleic acid moleculedescribed herein targets a splice site of exon 8, 23, 35, 43, 44, 45,50, 51, 52, 53, or 55 of the DMD gene. In some cases, a polynucleic acidmolecule described herein targets a splice site of exon 8 of the DMDgene. In some instances, a polynucleic acid molecule described hereintargets a splice site of exon 23 of the DMD gene. In some cases, apolynucleic acid molecule described herein targets a splice site of exon35 of the DMD gene. In some cases, a polynucleic acid molecule describedherein targets a splice site of exon 43 of the DMD gene. In some cases,a polynucleic acid molecule described herein targets a splice site ofexon 44 of the DMD gene. In some cases, a polynucleic acid moleculedescribed herein targets a splice site of exon 45 of the DMD gene. Insome instances, a polynucleic acid molecule described herein targets asplice site of exon 48 of the DMD gene. In some instances, a polynucleicacid molecule described herein targets a splice site of exon 49 of theDMD gene. In some instances, a polynucleic acid molecule describedherein targets a splice site of exon 50 of the DMD gene. In someinstances, a polynucleic acid molecule described herein targets a splicesite of exon 51 of the DMD gene. In some cases, a polynucleic acidmolecule described herein targets a splice site of exon 52 of the DMDgene. In some cases, a polynucleic acid molecule described hereintargets a splice site of exon 53 of the DMD gene. In some cases, apolynucleic acid molecule described herein targets a splice site of exon55 of the DMD gene. As used herein, a splice site includes a canonicalsplice site, a cryptic splice site or an alternative splice site that iscapable of inducing an insertion, deletion, duplication, or alterationin an incorrectly spliced mRNA transcript to induce exon skipping orexon inclusion.

In some embodiments, a polynucleic acid molecule described herein targeta partially spliced mRNA sequence comprising additional exons involvedin Duchenne muscular dystrophy such as exon 3, 4, 5, 6, 7, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, or 63.

In some instances, a polynucleic acid molecule described herein targetsa region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt,100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 ntupstream (or from the 5′) of exon 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, or 63 of the DMD gene. In some instances, a polynucleic acidmolecule described herein targets a region at least 1000 nt, 500 nt, 400nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10nt, or 5 nt upstream (or from the 5′) of exon 8, 23, 35, 43, 44, 45, 50,51, 52, 53, or 55 of the DMD gene. In some instances, a polynucleic acidmolecule described herein targets a region at least 1000 nt, 500 nt, 400nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10nt, or 5 nt upstream (or from the 5′) of exon 8 of the DMD gene. In someinstances, a polynucleic acid molecule described herein targets a regionat least 1000 nt, 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt,50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt upstream (or from the 5′) ofexon 23 of the DMD gene. In some instances, a polynucleic acid moleculedescribed herein targets a region at least 1000 nt, 500 nt, 400 nt, 300nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or5 nt upstream (or from the 5′) of exon 35 of the DMD gene. In someinstances, a polynucleic acid molecule described herein targets a regionat least 1000 nt, 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt,50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt upstream (or from the 5′) ofexon 43 of the DMD gene. In some instances, a polynucleic acid moleculedescribed herein targets a region at least 1000 nt, 500 nt, 400 nt, 300nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or5 nt upstream (or from the 5′) of exon 44 of the DMD gene. In someinstances, a polynucleic acid molecule described herein targets a regionat least 1000 nt, 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt,50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt upstream (or from the 5′) ofexon 45 of the DMD gene. In some instances, a polynucleic acid moleculedescribed herein targets a region at least 1000 nucleotides (nt), 500nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt,20 nt, 10 nt, or 5 nt upstream (or from the 5′) of exon 48 of the DMDgene. In some instances, a polynucleic acid molecule described hereintargets a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt,200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 ntupstream (or from the 5′) of exon 49 of the DMD gene. In some instances,a polynucleic acid molecule described herein targets a region at least1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt upstream (or from the 5′)of exon 50 of the DMD gene. In some instances, a polynucleic acidmolecule described herein targets a region at least 1000 nucleotides(nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40nt, 30 nt, 20 nt, 10 nt, or 5 nt upstream (or from the 5′) of exon 51 ofthe DMD gene. In some instances, a polynucleic acid molecule describedherein targets a region at least 1000 nucleotides (nt), 500 nt, 400 nt,300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt,or 5 nt upstream (or from the 5′) of exon 52 of the DMD gene. In someinstances, a polynucleic acid molecule described herein targets a regionat least 1000 nt, 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt,50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt upstream (or from the 5′) ofexon 53 of the DMD gene. In some instances, a polynucleic acid moleculedescribed herein targets a region at least 1000 nt, 500 nt, 400 nt, 300nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or5 nt upstream (or from the 5′) of exon 55 of the DMD gene.

In some instances, the polynucleic acid molecule hybridizes to a targetregion that is upstream (or 5′) to at least one of exon 3, 4, 5, 6, 7,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene. In someinstances, the polynucleic acid molecule hybridizes to a target regionthat is upstream (or 5′) to at least one of exon 8, 23, 35, 43, 44, 45,50, 51, 52, 53, or 55 of the DMD gene. In some instances, thepolynucleic acid molecule hybridizes to a target region that is about 5,10, 15, 20, 50, 100, 200, 300, 400 or 500 bp upstream (or 5′) to atleast one of exon 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63of the DMD gene.

In some instances, a polynucleic acid molecule described herein targetsa region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt,100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 ntdownstream (or from the 3′) of exon 3, 4, 5, 6, 7, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, or 63 of the DMD gene. In some instances, a polynucleicacid molecule described herein targets a region at least 1000nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt,50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or from the 3′)of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. Insome instances, a polynucleic acid molecule described herein targets aregion at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt,100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 ntdownstream (or from the 3′) of exon 8 of the DMD gene. In someinstances, a polynucleic acid molecule described herein targets a regionat least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt,80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (orfrom the 3′) of exon 23 of the DMD gene. In some instances, apolynucleic acid molecule described herein targets a region at least1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or from the3′) of exon 35 of the DMD gene. In some instances, a polynucleic acidmolecule described herein targets a region at least 1000 nucleotides(nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or from the 3′) of exon 43of the DMD gene. In some instances, a polynucleic acid moleculedescribed herein targets a region at least 1000 nucleotides (nt), 500nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt,20 nt, 10 nt, or 5 nt downstream (or from the 3′) of exon 44 of the DMDgene. In some instances, a polynucleic acid molecule described hereintargets a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt,200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 ntdownstream (or from the 3′) of exon 45 of the DMD gene. In someinstances, a polynucleic acid molecule described herein targets a regionat least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt,80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (orfrom the 3′) of exon 48 of the DMD gene. In some instances, apolynucleic acid molecule described herein targets a region at least1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or from the3′) of exon 49 of the DMD gene. In some instances, a polynucleic acidmolecule described herein targets a region at least 1000 nucleotides(nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or from the 3′) of exon 50of the DMD gene. In some instances, a polynucleic acid moleculedescribed herein targets a region at least 1000 nucleotides (nt), 500nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt,20 nt, 10 nt, or 5 nt downstream (or from the 3′) of exon 51 of the DMDgene. In some instances, a polynucleic acid molecule described hereintargets a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt,200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 ntdownstream (or from the 3′) of exon 52 of the DMD gene. In someinstances, a polynucleic acid molecule described herein targets a regionat least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt,80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (orfrom the 3′) of exon 53 of the DMD gene. In some instances, apolynucleic acid molecule described herein targets a region at least1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or from the3′) of exon 55 of the DMD gene.

In some instances, the polynucleic acid molecule hybridizes to a targetregion that is downstream (or 3′) to at least one of exon 3, 4, 5, 6, 7,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene. In someinstances, the polynucleic acid molecule hybridizes to a target regionthat is about 5, 10, 15, 20, 50, 100, 200, 300, 400 or 500 bp downstream(or 3′) to at least one of exon 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, and 63 of the DMD gene. In some instances, the polynucleic acidmolecule hybridizes to a target region that is about 5, 10, 15, 20, 50,100, 200, 300, 400 or 500 bp downstream (or 3′) to at least one of exon8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene.

In some instances, a polynucleic acid molecule described herein targetsan internal region within exon 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, or 63 of the DMD gene. In some instances, a polynucleic acidmolecule described herein targets an internal region within exon 8, 23,35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In someinstances, a polynucleic acid molecule described herein targets aninternal region within exon 8 of the DMD gene. In some instances, apolynucleic acid molecule described herein targets an internal regionwithin exon 23 of the DMD gene. In some instances, a polynucleic acidmolecule described herein targets an internal region within exon 35 ofthe DMD gene. In some instances, a polynucleic acid molecule describedherein targets an internal region within exon 43 of the DMD gene. Insome instances, a polynucleic acid molecule described herein targets aninternal region within exon 44 of the DMD gene. In some instances, apolynucleic acid molecule described herein targets an internal regionwithin exon 45 of the DMD gene. In some instances, a polynucleic acidmolecule described herein targets an internal region within exon 48 ofthe DMD gene. In some instances, a polynucleic acid molecule describedherein targets an internal region within exon 49 of the DMD gene. Insome instances, a polynucleic acid molecule described herein targets aninternal region within exon 50 of the DMD gene. In some instances, apolynucleic acid molecule described herein targets an internal regionwithin exon 51 of the DMD gene. In some instances, a polynucleic acidmolecule described herein targets an internal region within exon 52 ofthe DMD gene. In some instances, a polynucleic acid molecule describedherein targets an internal region within exon 53 of the DMD gene. Insome instances, a polynucleic acid molecule described herein targets aninternal region within exon 55 of the DMD gene.

In some instances, the polynucleic acid molecule hybridizes to a targetregion that is within at least one of exon 3, 4, 5, 6, 7, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, and 63 of the DMD gene. In some instances, thepolynucleic acid molecule hybridizes to a target region that is withinat least one of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of theDMD gene.

In some embodiments, a polynucleic acid molecule described hereintargets a partially spliced mRNA sequence comprising exon 51. In someinstances, the polynucleic acid molecule hybridizes to a target regionthat is upstream (or 5′) to exon 51. In some instances, the polynucleicacid molecule hybridizes to a target region that is about 5, 10, 15, 20,50, 100, 200, 300, 400 or 500 bp upstream (or 5′) to exon 51. In someinstances, the polynucleic acid molecule hybridizes to a target regionthat is downstream (or 3′) to exon 51. In some instances, thepolynucleic acid molecule hybridizes to a target region that is about 5,10, 15, 20, 50, 100, 200, 300, 400 or 500 bp downstream (or 3′) to exon51.

In some instances, the polynucleic acid molecule hybridizes to a targetregion that is within exon 51. In some instances, the polynucleic acidmolecule hybridizes to a target region that is at either the 5′intron-exon 51 junction or the 3′ exon 51-intron junction.

In some embodiments, the polynucleic acid molecule comprises a sequencehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to a target sequence ofinterest. In some embodiments, the polynucleic acid molecule comprises asequence having at least 50% sequence identity to a target sequence ofinterest. In some embodiments, the polynucleic acid molecule comprises asequence having at least 60% sequence identity to a target sequence ofinterest. In some embodiments, the polynucleic acid molecule comprises asequence having at least 70% sequence identity to a target sequence ofinterest. In some embodiments, the polynucleic acid molecule comprises asequence having at least 75% sequence identity to a target sequence ofinterest. In some embodiments, the polynucleic acid molecule comprises asequence having at least 80% sequence identity to a target sequence ofinterest. In some embodiments, the polynucleic acid molecule comprises asequence having at least 85% sequence identity to a target sequence ofinterest. In some embodiments, the polynucleic acid molecule comprises asequence having at least 90% sequence identity to a target sequence ofinterest. In some embodiments, the polynucleic acid molecule comprises asequence having at least 95% sequence identity to a target sequence ofinterest. In some embodiments, the polynucleic acid molecule comprises asequence having at least 96% sequence identity to a target sequence ofinterest. In some embodiments, the polynucleic acid molecule comprises asequence having at least 97% sequence identity to a target sequence ofinterest. In some embodiments, the polynucleic acid molecule comprises asequence having at least 98% sequence identity to a target sequence ofinterest. In some embodiments, the polynucleic acid molecule comprises asequence having at least 99% sequence identity to a target sequence ofinterest. In some embodiments, the polynucleic acid molecule consists ofa target sequence of interest.

In some embodiments, the polynucleic acid molecule comprises a firstpolynucleotide and a second polynucleotide. In some instances, the firstpolynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a target sequence of interest. In some cases, the secondpolynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a target sequence of interest. In some cases, thepolynucleic acid molecule comprises a first polynucleotide having atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to a target sequence of interest and asecond polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a targetsequence of interest.

In some embodiments, the polynucleic acid molecule described hereincomprises RNA or DNA. In some cases, the polynucleic acid moleculecomprises RNA. In some instances, RNA comprises short interfering RNA(siRNA), short hairpin RNA (shRNA), microRNA (miRNA), double-strandedRNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heterogeneousnuclear RNA (hnRNA). In some instances, RNA comprises shRNA. In someinstances, RNA comprises miRNA. In some instances, RNA comprises dsRNA.In some instances, RNA comprises tRNA. In some instances, RNA comprisesrRNA. In some instances, RNA comprises hnRNA. In some instances, the RNAcomprises siRNA. In some instances, the polynucleic acid moleculecomprises siRNA.

In some embodiments, the polynucleic acid molecule is from about 10 toabout 50 nucleotides in length. In some instances, the polynucleic acidmolecule is from about 10 to about 30, from about 15 to about 30, fromabout 18 to about 25, form about 18 to about 24, from about 19 to about23, or from about 20 to about 22 nucleotides in length.

In some embodiments, the polynucleic acid molecule is about 50nucleotides in length. In some instances, the polynucleic acid moleculeis about 45 nucleotides in length. In some instances, the polynucleicacid molecule is about 40 nucleotides in length. In some instances, thepolynucleic acid molecule is about 35 nucleotides in length. In someinstances, the polynucleic acid molecule is about 30 nucleotides inlength. In some instances, the polynucleic acid molecule is about 25nucleotides in length. In some instances, the polynucleic acid moleculeis about 20 nucleotides in length. In some instances, the polynucleicacid molecule is about 19 nucleotides in length. In some instances, thepolynucleic acid molecule is about 18 nucleotides in length. In someinstances, the polynucleic acid molecule is about 17 nucleotides inlength. In some instances, the polynucleic acid molecule is about 16nucleotides in length. In some instances, the polynucleic acid moleculeis about 15 nucleotides in length. In some instances, the polynucleicacid molecule is about 14 nucleotides in length. In some instances, thepolynucleic acid molecule is about 13 nucleotides in length. In someinstances, the polynucleic acid molecule is about 12 nucleotides inlength. In some instances, the polynucleic acid molecule is about 11nucleotides in length. In some instances, the polynucleic acid moleculeis about 10 nucleotides in length. In some instances, the polynucleicacid molecule is between about 10 and about 50 nucleotides in length. Insome instances, the polynucleic acid molecule is between about 10 andabout 45 nucleotides in length. In some instances, the polynucleic acidmolecule is between about 10 and about 40 nucleotides in length. In someinstances, the polynucleic acid molecule is between about 10 and about35 nucleotides in length. In some instances, the polynucleic acidmolecule is between about 10 and about 30 nucleotides in length. In someinstances, the polynucleic acid molecule is between about 10 and about25 nucleotides in length. In some instances, the polynucleic acidmolecule is between about 10 and about 20 nucleotides in length. In someinstances, the polynucleic acid molecule is between about 15 and about25 nucleotides in length. In some instances, the polynucleic acidmolecule is between about 15 and about 30 nucleotides in length. In someinstances, the polynucleic acid molecule is between about 12 and about30 nucleotides in length.

In some embodiments, the polynucleic acid molecule comprises a firstpolynucleotide. In some instances, the polynucleic acid moleculecomprises a second polynucleotide. In some instances, the polynucleicacid molecule comprises a first polynucleotide and a secondpolynucleotide. In some instances, the first polynucleotide is a sensestrand or passenger strand. In some instances, the second polynucleotideis an antisense strand or guide strand.

In some embodiments, the polynucleic acid molecule is a firstpolynucleotide. In some embodiments, the first polynucleotide is fromabout 10 to about 50 nucleotides in length. In some instances, the firstpolynucleotide is from about 10 to about 30, from about 15 to about 30,from about 18 to about 25, form about 18 to about 24, from about 19 toabout 23, or from about 20 to about 22 nucleotides in length.

In some instances, the first polynucleotide is about 50 nucleotides inlength. In some instances, the first polynucleotide is about 45nucleotides in length. In some instances, the first polynucleotide isabout 40 nucleotides in length. In some instances, the firstpolynucleotide is about 35 nucleotides in length. In some instances, thefirst polynucleotide is about 30 nucleotides in length. In someinstances, the first polynucleotide is about 25 nucleotides in length.In some instances, the first polynucleotide is about 20 nucleotides inlength. In some instances, the first polynucleotide is about 19nucleotides in length. In some instances, the first polynucleotide isabout 18 nucleotides in length. In some instances, the firstpolynucleotide is about 17 nucleotides in length. In some instances, thefirst polynucleotide is about 16 nucleotides in length. In someinstances, the first polynucleotide is about 15 nucleotides in length.In some instances, the first polynucleotide is about 14 nucleotides inlength. In some instances, the first polynucleotide is about 13nucleotides in length. In some instances, the first polynucleotide isabout 12 nucleotides in length. In some instances, the firstpolynucleotide is about 11 nucleotides in length. In some instances, thefirst polynucleotide is about 10 nucleotides in length. In someinstances, the first polynucleotide is between about 10 and about 50nucleotides in length. In some instances, the first polynucleotide isbetween about 10 and about 45 nucleotides in length. In some instances,the first polynucleotide is between about 10 and about 40 nucleotides inlength. In some instances, the first polynucleotide is between about 10and about 35 nucleotides in length. In some instances, the firstpolynucleotide is between about 10 and about 30 nucleotides in length.In some instances, the first polynucleotide is between about 10 andabout 25 nucleotides in length. In some instances, the firstpolynucleotide is between about 10 and about 20 nucleotides in length.In some instances, the first polynucleotide is between about 15 andabout 25 nucleotides in length. In some instances, the firstpolynucleotide is between about 15 and about 30 nucleotides in length.In some instances, the first polynucleotide is between about 12 andabout 30 nucleotides in length.

In some embodiments, the polynucleic acid molecule is a secondpolynucleotide. In some embodiments, the second polynucleotide is fromabout 10 to about 50 nucleotides in length. In some instances, thesecond polynucleotide is from about 10 to about 30, from about 15 toabout 30, from about 18 to about 25, form about 18 to about 24, fromabout 19 to about 23, or from about 20 to about 22 nucleotides inlength.

In some instances, the second polynucleotide is about 50 nucleotides inlength. In some instances, the second polynucleotide is about 45nucleotides in length. In some instances, the second polynucleotide isabout 40 nucleotides in length. In some instances, the secondpolynucleotide is about 35 nucleotides in length. In some instances, thesecond polynucleotide is about 30 nucleotides in length. In someinstances, the second polynucleotide is about 25 nucleotides in length.In some instances, the second polynucleotide is about 20 nucleotides inlength. In some instances, the second polynucleotide is about 19nucleotides in length. In some instances, the second polynucleotide isabout 18 nucleotides in length. In some instances, the secondpolynucleotide is about 17 nucleotides in length. In some instances, thesecond polynucleotide is about 16 nucleotides in length. In someinstances, the second polynucleotide is about 15 nucleotides in length.In some instances, the second polynucleotide is about 14 nucleotides inlength. In some instances, the second polynucleotide is about 13nucleotides in length. In some instances, the second polynucleotide isabout 12 nucleotides in length. In some instances, the secondpolynucleotide is about 11 nucleotides in length. In some instances, thesecond polynucleotide is about 10 nucleotides in length. In someinstances, the second polynucleotide is between about 10 and about 50nucleotides in length. In some instances, the second polynucleotide isbetween about 10 and about 45 nucleotides in length. In some instances,the second polynucleotide is between about 10 and about 40 nucleotidesin length. In some instances, the second polynucleotide is between about10 and about 35 nucleotides in length. In some instances, the secondpolynucleotide is between about 10 and about 30 nucleotides in length.In some instances, the second polynucleotide is between about 10 andabout 25 nucleotides in length. In some instances, the secondpolynucleotide is between about 10 and about 20 nucleotides in length.In some instances, the second polynucleotide is between about 15 andabout 25 nucleotides in length. In some instances, the secondpolynucleotide is between about 15 and about 30 nucleotides in length.In some instances, the second polynucleotide is between about 12 andabout 30 nucleotides in length.

In some embodiments, the polynucleic acid molecule comprises a firstpolynucleotide and a second polynucleotide. In some instances, thepolynucleic acid molecule further comprises a blunt terminus, anoverhang, or a combination thereof. In some instances, the bluntterminus is a 5′ blunt terminus, a 3′ blunt terminus, or both. In somecases, the overhang is a 5′ overhang, 3′ overhang, or both. In somecases, the overhang comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-basepairing nucleotides. In some cases, the overhang comprises 1, 2, 3, 4,5, or 6 non-base pairing nucleotides. In some cases, the overhangcomprises 1, 2, 3, or 4 non-base pairing nucleotides. In some cases, theoverhang comprises 1 non-base pairing nucleotide. In some cases, theoverhang comprises 2 non-base pairing nucleotides. In some cases, theoverhang comprises 3 non-base pairing nucleotides. In some cases, theoverhang comprises 4 non-base pairing nucleotides.

In some embodiments, the sequence of the polynucleic acid molecule is atleast 40%, 50%, 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, 95% 98%, 99%, or99.5% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least50% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least60% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least70% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least80% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least90% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least95% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least99% complementary to a target sequence described herein. In someinstances, the sequence of the polynucleic acid molecule is 100%complementary to a target sequence described herein.

In some embodiments, the sequence of the polynucleic acid molecule has 5or less mismatches to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule has 4 or lessmismatches to a target sequence described herein. In some instances, thesequence of the polynucleic acid molecule has 3 or less mismatches to atarget sequence described herein. In some cases, the sequence of thepolynucleic acid molecule has 2 or less mismatches to a target sequencedescribed herein. In some cases, the sequence of the polynucleic acidmolecule has 1 or less mismatches to a target sequence described herein.

In some embodiments, the specificity of the polynucleic acid moleculethat hybridizes to a target sequence described herein is a 95%, 98%,99%, 99.5% or 100% sequence complementarity of the polynucleic acidmolecule to a target sequence. In some instances, the hybridization is ahigh stringent hybridization condition.

In some embodiments, the polynucleic acid molecule has reducedoff-target effect. In some instances, “off-target” or “off-targeteffects” refer to any instance in which a polynucleic acid polymerdirected against a given target causes an unintended effect byinteracting either directly or indirectly with another mRNA sequence, aDNA sequence or a cellular protein or other moiety. In some instances,an “off-target effect” occurs when there is a simultaneous degradationof other transcripts due to partial homology or complementarity betweenthat other transcript and the sense and/or antisense strand of thepolynucleic acid molecule.

In some embodiments, the polynucleic acid molecule comprises natural orsynthetic or artificial nucleotide analogues or bases. In some cases,the polynucleic acid molecule comprises combinations of DNA, RNA and/ornucleotide analogues. In some instances, the synthetic or artificialnucleotide analogues or bases comprise modifications at one or more ofribose moiety, phosphate moiety, nucleoside moiety, or a combinationthereof.

In some embodiments, nucleotide analogues or artificial nucleotide basecomprise a nucleic acid with a modification at a 2′ hydroxyl group ofthe ribose moiety. In some instances, the modification includes an H,OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety.Exemplary alkyl moiety includes, but is not limited to, halogens,sulfurs, thiols, thioethers, thioesters, amines (primary, secondary, ortertiary), amides, ethers, esters, alcohols and oxygen. In someinstances, the alkyl moiety further comprises a modification. In someinstances, the modification comprises an azo group, a keto group, analdehyde group, a carboxyl group, a nitro group, a nitroso, group, anitrile group, a heterocycle (e.g., imidazole, hydrazino orhydroxylamino) group, an isocyanate or cyanate group, or a sulfurcontaining group (e.g., sulfoxide, sulfone, sulfide, and disulfide). Insome instances, the alkyl moiety further comprises a heterosubstitution. In some instances, the carbon of the heterocyclic group issubstituted by a nitrogen, oxygen or sulfur. In some instances, theheterocyclic substitution includes but is not limited to, morpholino,imidazole, and pyrrolidino.

In some instances, the modification at the 2′ hydroxyl group is a2′-O-methyl modification or a 2′-O-methoxyethyl (2′-O-MOE) modification.In some cases, the 2′-O-methyl modification adds a methyl group to the2′ hydroxyl group of the ribose moiety whereas the 2′O-methoxyethylmodification adds a methoxyethyl group to the 2′ hydroxyl group of theribose moiety. Exemplary chemical structures of a 2′-O-methylmodification of an adenosine molecule and 2′O-methoxyethyl modificationof an uridine are illustrated below.

In some instances, the modification at the 2′ hydroxyl group is a2′-O-aminopropyl modification in which an extended amine groupcomprising a propyl linker binds the amine group to the 2′ oxygen. Insome instances, this modification neutralizes the phosphate derivedoverall negative charge of the oligonucleotide molecule by introducingone positive charge from the amine group per sugar and thereby improvescellular uptake properties due to its zwitterionic properties. Anexemplary chemical structure of a 2′-O-aminopropyl nucleosidephosphoramidite is illustrated below.

In some instances, the modification at the 2′ hydroxyl group is a lockedor bridged ribose modification (e.g., locked nucleic acid or LNA) inwhich the oxygen molecule bound at the 2′ carbon is linked to the 4′carbon by a methylene group, thus forming a2′-C,4′-C-oxy-methylene-linked bicyclic ribonucleotide monomer.Exemplary representations of the chemical structure of LNA areillustrated below. The representation shown to the left highlights thechemical connectivities of an LNA monomer. The representation shown tothe right highlights the locked 3′-endo (³E) conformation of thefuranose ring of an LNA monomer.

In some instances, the modification at the 2′ hydroxyl group comprisesethylene nucleic acids (ENA) such as for example 2′-4′-ethylene-bridgednucleic acid, which locks the sugar conformation into a C3′-endo sugarpuckering conformation. ENA are part of the bridged nucleic acids classof modified nucleic acids that also comprises LNA. Exemplary chemicalstructures of the ENA and bridged nucleic acids are illustrated below.

In some embodiments, additional modifications at the 2′ hydroxyl groupinclude 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA).

In some embodiments, nucleotide analogues comprise modified bases suchas, but not limited to, 5-propynyluridine, 5-propynylcytidine,6-methyladenine, 6-methylguanine, N, N, -dimethyladenine,2-propyladenine, 2propylguanine, 2-aminoadenine, 1-methylinosine,3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotideshaving a modification at the 5 position, 5-(2-amino) propyl uridine,5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine,2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine,7-methylguanosine, 2, 2-dimethylguanosine, 5-methylaminoethyluridine,5-methyloxyuridine, deazanucleotides such as 7-deaza-adenosine,6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiouridine,other thio bases such as 2-thiouridine and 4-thiouridine and2-thiocytidine, dihydrouridine, pseudouridine, queuosine, archaeosine,naphthyl and substituted naphthyl groups, any O- and N-alkylated purinesand pyrimidines such as N6-methyladenosine,5-methylcarbonylmethyluridine, uridine 5-oxy acetic acid,pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groups suchas aminophenol or 2,4,6-trimethoxy benzene, modified cytosines that actas G-clamp nucleotides, 8-substituted adenines and guanines,5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkylnucleotides, carboxyalkylaminoalkyi nucleotides, andalkylcarbonylalkylated nucleotides. Modified nucleotides also includethose nucleotides that are modified with respect to the sugar moiety, aswell as nucleotides having sugars or analogs thereof that are notribosyl. For example, the sugar moieties, in some cases are or be basedon, mannoses, arabinoses, glucopyranoses, galactopyranoses,4′-thioribose, and other sugars, heterocycles, or carbocycles. The termnucleotide also includes what are known in the art as universal bases.By way of example, universal bases include but are not limited to3-nitropyrrole, 5-nitroindole, or nebularine.

In some embodiments, nucleotide analogues further comprise morpholinos,peptide nucleic acids (PNAs), methylphosphonate nucleotides,thiolphosphonate nucleotides, 2′-fluoro N3-P5′-phosphoramidites, 1′,5′-anhydrohexitol nucleic acids (HNAs), or a combination thereof.Morpholino or phosphorodiamidate morpholino oligo (PMO) comprisessynthetic molecules whose structure mimics natural nucleic acidstructure by deviates from the normal sugar and phosphate structures. Insome instances, the five member ribose ring is substituted with a sixmember morpholino ring containing four carbons, one nitrogen and oneoxygen. In some cases, the ribose monomers are linked by aphosphordiamidate group instead of a phosphate group. In such cases, thebackbone alterations remove all positive and negative charges makingmorpholinos neutral molecules capable of crossing cellular membraneswithout the aid of cellular delivery agents such as those used bycharged oligonucleotides.

In some embodiments, peptide nucleic acid (PNA) does not contain sugarring or phosphate linkage and the bases are attached and appropriatelyspaced by oligoglycine-like molecules, therefore, eliminating a backbonecharge.

In some embodiments, one or more modifications optionally occur at theinternucleotide linkage. In some instances, modified internucleotidelinkage include, but is not limited to, phosphorothioates,phosphorodithioates, methylphosphonates, 5′-alkylenephosphonates,5′-methylphosphonate, 3′-alkylene phosphonates, borontrifluoridates,borano phosphate esters and selenophosphates of 3′-5′linkage or2′-5′linkage, phosphotriesters, thionoalkylphosphotriesters, hydrogenphosphonate linkages, alkyl phosphonates, alkylphosphonothioates,arylphosphonothioates, phosphoroselenoates, phosphorodiselenoates,phosphinates, phosphoramidates, 3′-alkylphosphoramidates,aminoalkylphosphoramidates, thionophosphoramidates,phosphoropiperazidates, phosphoroanilothioates, phosphoroanilidates,ketones, sulfones, sulfonamides, carbonates, carbamates,methylenehydrazos, methylenedimethylhydrazos, formacetals,thioformacetals, oximes, methyleneiminos, methylenemethyliminos,thioamidates, linkages with riboacetyl groups, aminoethyl glycine, silylor siloxane linkages, alkyl or cycloalkyl linkages with or withoutheteroatoms of, for example, 1 to 10 carbons that are saturated orunsaturated and/or substituted and/or contain heteroatoms, linkages withmorpholino structures, amides, polyamides wherein the bases are attachedto the aza nitrogens of the backbone directly or indirectly, andcombinations thereof. Phosphorothioate antisene oligonucleotides (PSASO) are antisense oligonucleotides comprising a phosphorothioatelinkage. An exemplary PS ASO is illustrated below.

In some instances, the modification is a methyl or thiol modificationsuch as methylphosphonate or thiolphosphonate modification. Exemplarythiolphosphonate nucleotide (left) and methylphosphonate nucleotide(right) are illustrated below.

In some instances, a modified nucleotide includes, but is not limitedto, 2′-fluoro N3-P5′-phosphoramidites illustrated as:

In some instances, a modified nucleotide includes, but is not limitedto, hexitol nucleic acid (or 1′, 5′-anhydrohexitol nucleic acids (HNA))illustrated as:

In some embodiments, one or more modifications further optionallyinclude modifications of the ribose moiety, phosphate backbone and thenucleoside, or modifications of the nucleotide analogues at the 3′ orthe 5′ terminus. For example, the 3′ terminus optionally include a 3cationic group, or by inverting the nucleoside at the 3′-terminus with a3′-3 linkage. In another alternative, the 3′-terminus is optionallyconjugated with an aminoalkyl group, e.g., a 3′ C5-aminoalkyl dT. In anadditional alternative, the 3′-terminus is optionally conjugated with anabasic site, e.g., with an apurinic or apyrimidinic site. In someinstances, the 5-terminus is conjugated with an aminoalkyl group, e.g.,a 5′-O-alkylamino substituent. In some cases, the 5′-terminus isconjugated with an abasic site, e.g., with an apurinic or apyrimidinicsite.

In some embodiments, the polynucleic acid molecule comprises one or moreof the artificial nucleotide analogues described herein. In someinstances, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of theartificial nucleotide analogues described herein. In some embodiments,the artificial nucleotide analogues include 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonatenucleotides, thiolphosphonate nucleotides, 2′-fluoroN3-P5′-phosphoramidites, or a combination thereof. In some instances,the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of the artificialnucleotide analogues selected from 2′-O-methyl, 2′-O-methoxyethyl(2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro,2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified, LNA, ENA,PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonatenucleotides, 2′-fluoro N3-P5′-phosphoramidites, or a combinationthereof. In some instances, the polynucleic acid molecule comprises 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, ormore of 2′-O-methyl modified nucleotides. In some instances, thepolynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 20, 25, or more of 2′-O-methoxyethyl(2′-O-MOE) modified nucleotides. In some instances, the polynucleic acidmolecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 20, 25, or more of thiolphosphonate nucleotides.

In some instances, the polynucleic acid molecule comprises at least oneof: from about 5% to about 100% modification, from about 10% to about100% modification, from about 20% to about 100% modification, from about30% to about 100% modification, from about 40% to about 100%modification, from about 50% to about 100% modification, from about 60%to about 100% modification, from about 70% to about 100% modification,from about 80% to about 100% modification, and from about 90% to about100% modification.

In some cases, the polynucleic acid molecule comprises at least one of:from about 10% to about 90% modification, from about 20% to about 90%modification, from about 30% to about 90% modification, from about 40%to about 90% modification, from about 50% to about 90% modification,from about 60% to about 90% modification, from about 70% to about 90%modification, and from about 80% to about 100% modification.

In some cases, the polynucleic acid molecule comprises at least one of:from about 10% to about 80% modification, from about 20% to about 80%modification, from about 30% to about 80% modification, from about 40%to about 80% modification, from about 50% to about 80% modification,from about 60% to about 80% modification, and from about 70% to about80% modification.

In some instances, the polynucleic acid molecule comprises at least oneof: from about 10% to about 70% modification, from about 20% to about70% modification, from about 30% to about 70% modification, from about40% to about 70% modification, from about 50% to about 70% modification,and from about 60% to about 70% modification.

In some instances, the polynucleic acid molecule comprises at least oneof: from about 10% to about 60% modification, from about 20% to about60% modification, from about 30% to about 60% modification, from about40% to about 60% modification, and from about 50% to about 60%modification.

In some cases, the polynucleic acid molecule comprises at least one of:from about 10% to about 50% modification, from about 20% to about 50%modification, from about 30% to about 50% modification, and from about40% to about 50% modification.

In some cases, the polynucleic acid molecule comprises at least one of:from about 10% to about 40% modification, from about 20% to about 40%modification, and from about 30% to about 40% modification.

In some cases, the polynucleic acid molecule comprises at least one of:from about 10% to about 30% modification, and from about 20% to about30% modification.

In some cases, the polynucleic acid molecule comprises from about 10% toabout 20% modification.

In some cases, the polynucleic acid molecule comprises from about 15% toabout 90%, from about 20% to about 80%, from about 30% to about 70%, orfrom about 40% to about 60% modifications.

In additional cases, the polynucleic acid molecule comprises at leastabout 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%modification.

In some embodiments, the polynucleic acid molecule comprises at leastabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22 ormore modifications.

In some instances, the polynucleic acid molecule comprises at leastabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22 ormore modified nucleotides.

In some instances, from about 5% to about 100% of the polynucleic acidmolecule comprise the artificial nucleotide analogues described herein.In some instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of thepolynucleic acid molecule comprise the artificial nucleotide analoguesdescribed herein. In some instances, about 5% of a polynucleic acidmolecule comprise the artificial nucleotide analogues described herein.In some instances, about 10% of a polynucleic acid molecule comprise theartificial nucleotide analogues described herein. In some instances,about 15% of a polynucleic acid molecule comprise the artificialnucleotide analogues described herein. In some instances, about 20% of apolynucleic acid molecule comprise the artificial nucleotide analoguesdescribed herein. In some instances, about 25% of a polynucleic acidmolecule comprise the artificial nucleotide analogues described herein.In some instances, about 30% of a polynucleic acid molecule comprise theartificial nucleotide analogues described herein. In some instances,about 35% of a polynucleic acid molecule comprise the artificialnucleotide analogues described herein. In some instances, about 40% of apolynucleic acid molecule comprise the artificial nucleotide analoguesdescribed herein. In some instances, about 45% of a polynucleic acidmolecule comprise the artificial nucleotide analogues described herein.In some instances, about 50% of a polynucleic acid molecule comprise theartificial nucleotide analogues described herein. In some instances,about 55% of a polynucleic acid molecule comprise the artificialnucleotide analogues described herein. In some instances, about 60% of apolynucleic acid molecule comprise the artificial nucleotide analoguesdescribed herein. In some instances, about 65% of a polynucleic acidmolecule comprise the artificial nucleotide analogues described herein.In some instances, about 70% of a polynucleic acid molecule comprise theartificial nucleotide analogues described herein. In some instances,about 75% of a polynucleic acid molecule comprise the artificialnucleotide analogues described herein. In some instances, about 80% of apolynucleic acid molecule comprise the artificial nucleotide analoguesdescribed herein. In some instances, about 85% of a polynucleic acidmolecule comprise the artificial nucleotide analogues described herein.In some instances, about 90% of a polynucleic acid molecule comprise theartificial nucleotide analogues described herein. In some instances,about 95% of a polynucleic acid molecule comprise the artificialnucleotide analogues described herein. In some instances, about 96% of apolynucleic acid molecule comprise the artificial nucleotide analoguesdescribed herein. In some instances, about 97% of a polynucleic acidmolecule comprise the artificial nucleotide analogues described herein.In some instances, about 98% of a polynucleic acid molecule comprise theartificial nucleotide analogues described herein. In some instances,about 99% of a polynucleic acid molecule comprise the artificialnucleotide analogues described herein. In some instances, about 100% ofa polynucleic acid molecule comprise the artificial nucleotide analoguesdescribed herein. In some embodiments, the artificial nucleotideanalogues include 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl(2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified, LNA, ENA,PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonatenucleotides, 2′-fluoro N3-P5′-phosphoramidites, or a combinationthereof.

In some embodiments, the polynucleic acid molecule comprises from about1 to about 25 modifications in which the modification comprises anartificial nucleotide analogues described herein. In some embodiments, apolynucleic acid molecule comprises about 1 modification in which themodification comprises an artificial nucleotide analogue describedherein. In some embodiments, a polynucleic acid molecule comprises about2 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, a polynucleicacid molecule comprises about 3 modifications in which the modificationscomprise an artificial nucleotide analogue described herein. In someembodiments, a polynucleic acid molecule comprises about 4 modificationsin which the modifications comprise an artificial nucleotide analoguedescribed herein. In some embodiments, a polynucleic acid moleculecomprises about 5 modifications in which the modifications comprise anartificial nucleotide analogue described herein. In some embodiments, apolynucleic acid molecule comprises about 6 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, a polynucleic acid molecule comprises about7 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, a polynucleicacid molecule comprises about 8 modifications in which the modificationscomprise an artificial nucleotide analogue described herein. In someembodiments, a polynucleic acid molecule comprises about 9 modificationsin which the modifications comprise an artificial nucleotide analoguedescribed herein. In some embodiments, a polynucleic acid moleculecomprises about 10 modifications in which the modifications comprise anartificial nucleotide analogue described herein. In some embodiments, apolynucleic acid molecule comprises about 11 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, a polynucleic acid molecule comprises about12 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, a polynucleicacid molecule comprises about 13 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, a polynucleic acid molecule comprises about14 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, a polynucleicacid molecule comprises about 15 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, a polynucleic acid molecule comprises about16 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, a polynucleicacid molecule comprises about 17 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, a polynucleic acid molecule comprises about18 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, a polynucleicacid molecule comprises about 19 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, a polynucleic acid molecule comprises about20 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, a polynucleicacid molecule comprises about 21 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, a polynucleic acid molecule comprises about22 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, a polynucleicacid molecule comprises about 23 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, a polynucleic acid molecule comprises about24 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, a polynucleicacid molecule comprises about 25 modifications in which themodifications comprise an artificial nucleotide analogue describedherein.

In some embodiments, a polynucleic acid molecule is assembled from twoseparate polynucleotides wherein one polynucleotide comprises the sensestrand and the second polynucleotide comprises the antisense strand ofthe polynucleic acid molecule. In other embodiments, the sense strand isconnected to the antisense strand via a linker molecule, which in someinstances is a polynucleotide linker or a non-nucleotide linker.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and antisense strand, wherein pyrimidine nucleotides in the sensestrand comprises 2′-O-methylpyrimidine nucleotides and purinenucleotides in the sense strand comprise 2′-deoxy purine nucleotides. Insome embodiments, a polynucleic acid molecule comprises a sense strandand antisense strand, wherein pyrimidine nucleotides present in thesense strand comprise 2′-deoxy-2′-fluoro pyrimidine nucleotides andwherein purine nucleotides present in the sense strand comprise 2′-deoxypurine nucleotides.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and antisense strand, wherein the pyrimidine nucleotides whenpresent in said antisense strand are 2′-deoxy-2′-fluoro pyrimidinenucleotides and the purine nucleotides when present in said antisensestrand are 2′-O-methyl purine nucleotides.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and antisense strand, wherein the pyrimidine nucleotides whenpresent in said antisense strand are 2′-deoxy-2′-fluoro pyrimidinenucleotides and wherein the purine nucleotides when present in saidantisense strand comprise 2′-deoxy-purine nucleotides.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and antisense strand, wherein the sense strand includes aterminal cap moiety at the 5′-end, the 3′-end, or both of the 5′ and 3′ends of the sense strand. In other embodiments, the terminal cap moietyis an inverted deoxy abasic moiety.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and an antisense strand, wherein the antisense strand comprises aphosphate backbone modification at the 3′ end of the antisense strand.In some instances, the phosphate backbone modification is aphosphorothioate.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and an antisense strand, wherein the antisense strand comprises aglyceryl modification at the 3′ end of the antisense strand.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and an antisense strand, in which the sense strand comprises oneor more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or more phosphorothioate internucleotidelinkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or about one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and in which the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the antisense strand. In otherembodiments, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more, pyrimidine nucleotides of the sense and/or antisense strandare chemically-modified with 2′-deoxy, 2′-O-methyl and/or2′-deoxy-2′-fluoro nucleotides, with or without one or more, for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′-end,the 5′-end, or both of the 3′- and 5′-ends, being present in the same ordifferent strand.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and an antisense strand, in which the sense strand comprisesabout 1 to about 25, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioateinternucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of thesense strand; and in which the antisense strand comprises about 1 toabout 25 or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioateinternucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe antisense strand. In other embodiments, one or more, for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides ofthe sense and/or antisense strand are chemically-modified with 2′-deoxy,2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without about1 to about 25 or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′-end,the 5′-end, or both of the 3′- and 5′-ends, being present in the same ordifferent strand.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and an antisense strand, in which the antisense strand comprisesone or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioate internucleotidelinkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In other embodiments, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, or more pyrimidine nucleotides of the sense and/or antisensestrand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or2′-deoxy-2′-fluoro nucleotides, with or without one or more, forexample, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends, being present in the same ordifferent strand.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and an antisense strand, in which the antisense strand comprisesabout 1 to about 25 or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioateinternucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe sense strand; and wherein the antisense strand comprises about 1 toabout 25 or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioateinternucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe antisense strand. In other embodiments, one or more, for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides ofthe sense and/or antisense strand are chemically-modified with 2′-deoxy,2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without about1 to about 5, for example about 1, 2, 3, 4, 5 or more phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′-end,the 5′-end, or both of the 3′- and 5′-ends, being present in the same ordifferent strand.

In some embodiments, a polynucleic acid molecule described herein is achemically-modified short interfering nucleic acid molecule having about1 to about 25, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate internucleotidelinkages in each strand of the polynucleic acid molecule.

In another embodiment, a polynucleic acid molecule described hereincomprises 2′-5′ internucleotide linkages. In some instances, the 2′-5′internucleotide linkage(s) is at the 3′-end, the 5′-end, or both of the3′- and 5′-ends of one or both sequence strands. In addition instances,the 2′-5′ internucleotide linkage(s) is present at various otherpositions within one or both sequence strands, for example, about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkageof a pyrimidine nucleotide in one or both strands of the polynucleicacid molecule comprise a 2′-5′ internucleotide linkage, or about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkageof a purine nucleotide in one or both strands of the polynucleic acidmolecule comprise a 2′-5′ internucleotide linkage.

In some embodiments, a polynucleic acid molecule is a single strandedpolynucleic acid molecule that mediates RNAi activity in a cell orreconstituted in vitro system, wherein the polynucleic acid moleculecomprises a single stranded polynucleotide having complementarity to atarget nucleic acid sequence, and wherein one or more pyrimidinenucleotides present in the polynucleic acid are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any purine nucleotides present in the polynucleic acid are2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are2′-deoxy purine nucleotides or alternately a plurality of purinenucleotides are 2′-deoxy purine nucleotides), and a terminal capmodification, that is optionally present at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the antisense sequence, the polynucleicacid molecule optionally further comprising about 1 to about 4 (e.g.,about 1, 2, 3, or 4) terminal 2′-deoxynucleotides at the 3′-end of thepolynucleic acid molecule, wherein the terminal nucleotides furthercomprise one or more (e.g., 1, 2, 3, or 4) phosphorothioateinternucleotide linkages, and wherein the polynucleic acid moleculeoptionally further comprises a terminal phosphate group, such as a5′-terminal phosphate group.

In some cases, one or more of the artificial nucleotide analoguesdescribed herein are resistant toward nucleases such as for exampleribonuclease such as RNase H, deoxyribonuclease such as DNase, orexonuclease such as 5′-3′ exonuclease and 3′-5′ exonuclease whencompared to natural polynucleic acid molecules. In some instances,artificial nucleotide analogues comprising 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonatenucleotides, thiolphosphonate nucleotides, 2′-fluoroN3-P5′-phosphoramidites, or combinations thereof are resistant towardnucleases such as for example ribonuclease such as RNase H,deoxyribonuclease such as DNase, or exonuclease such as 5′-3′exonuclease and 3′-5′ exonuclease. In some instances, 2′-O-methylmodified polynucleic acid molecule is nuclease resistance (e.g., RNaseH, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance). In someinstances, 2′O-methoxyethyl (2′-O-MOE) modified polynucleic acidmolecule is nuclease resistance (e.g., RNase H, DNase, 5′-3′ exonucleaseor 3′-5′ exonuclease resistance). In some instances, 2′-O-aminopropylmodified polynucleic acid molecule is nuclease resistance (e.g., RNaseH, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance). In someinstances, 2′-deoxy modified polynucleic acid molecule is nucleaseresistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). In some instances, T-deoxy-2′-fluoro modified polynucleicacid molecule is nuclease resistance (e.g., RNase H, DNase, 5′-3′exonuclease or 3′-5′ exonuclease resistance). In some instances,2′-O-aminopropyl (2′-O-AP) modified polynucleic acid molecule isnuclease resistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′exonuclease resistance). In some instances, 2′-O-dimethylaminoethyl(2′-O-DMAOE) modified polynucleic acid molecule is nuclease resistance(e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). In some instances, 2′-O-dimethylaminopropyl (2′-O-DMAP)modified polynucleic acid molecule is nuclease resistance (e.g., RNaseH, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance). In someinstances, T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE) modifiedpolynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase,5′-3′ exonuclease or 3′-5′ exonuclease resistance). In some instances,2′-O—N-methylacetamido (2′-O-NMA) modified polynucleic acid molecule isnuclease resistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′exonuclease resistance). In some instances, LNA modified polynucleicacid molecule is nuclease resistance (e.g., RNase H, DNase, 5′-3′exonuclease or 3′-5′ exonuclease resistance). In some instances, ENAmodified polynucleic acid molecule is nuclease resistance (e.g., RNaseH, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance). In someinstances, HNA modified polynucleic acid molecule is nuclease resistance(e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). In some instances, morpholinos is nuclease resistance(e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). In some instances, PNA modified polynucleic acid moleculeis resistant to nucleases (e.g., RNase H, DNase, 5′-3′ exonuclease or3′-5′ exonuclease resistance). In some instances, methylphosphonatenucleotides modified polynucleic acid molecule is nuclease resistance(e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). In some instances, thiolphosphonate nucleotides modifiedpolynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase,5′-3′ exonuclease or 3′-5′ exonuclease resistance). In some instances,polynucleic acid molecule comprising 2′-fluoro N3-P5′-phosphoramiditesis nuclease resistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′exonuclease resistance). In some instances, the 5′ conjugates describedherein inhibit 5′-3′ exonucleolytic cleavage. In some instances, the 3°conjugates described herein inhibit 3-5′ exonucleolytic cleavage.

In some embodiments, one or more of the artificial nucleotide analoguesdescribed herein have increased binding affinity toward their mRNAtarget relative to an equivalent natural polynucleic acid molecule. Theone or more of the artificial nucleotide analogues comprising2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonatenucleotides, thiolphosphonate nucleotides, or 2′-fluoroN3-P5′-phosphoramidites have increased binding affinity toward theirmRNA target relative to an equivalent natural polynucleic acid molecule.In some instances, 2′-O-methyl modified polynucleic acid molecule hasincreased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-methoxyethyl (2′-O-MOE) modified polynucleic acid molecule hasincreased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-aminopropyl modified polynucleic acid molecule has increasedbinding affinity toward their mRNA target relative to an equivalentnatural polynucleic acid molecule. In some instances, 2′-deoxy modifiedpolynucleic acid molecule has increased binding affinity toward theirmRNA target relative to an equivalent natural polynucleic acid molecule.In some instances, T-deoxy-2′-fluoro modified polynucleic acid moleculehas increased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-aminopropyl (2′-O-AP) modified polynucleic acid molecule hasincreased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-dimethylaminoethyl (2′-O-DMAOE) modified polynucleic acid moleculehas increased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-dimethylaminopropyl (2′-O-DMAP) modified polynucleic acid moleculehas increased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE) modified polynucleic acidmolecule has increased binding affinity toward their mRNA targetrelative to an equivalent natural polynucleic acid molecule. In someinstances, 2′-O—N-methylacetamido (2′-O-NMA) modified polynucleic acidmolecule has increased binding affinity toward their mRNA targetrelative to an equivalent natural polynucleic acid molecule. In someinstances, LNA modified polynucleic acid molecule has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some instances, ENA modified polynucleicacid molecule has increased binding affinity toward their mRNA targetrelative to an equivalent natural polynucleic acid molecule. In someinstances, PNA modified polynucleic acid molecule has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some instances, HNA modified polynucleicacid molecule has increased binding affinity toward their mRNA targetrelative to an equivalent natural polynucleic acid molecule. In someinstances, morpholino modified polynucleic acid molecule has increasedbinding affinity toward their mRNA target relative to an equivalentnatural polynucleic acid molecule. In some instances, methylphosphonatenucleotides modified polynucleic acid molecule has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some instances, thiolphosphonatenucleotides modified polynucleic acid molecule has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some instances, polynucleic acid moleculecomprising 2′-fluoro N3-P5′-phosphoramidites has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some cases, the increased affinity isillustrated with a lower Kd, a higher melt temperature (Tm), or acombination thereof.

In some embodiments, a polynucleic acid molecule described herein is achirally pure (or stereo pure) polynucleic acid molecule, or apolynucleic acid molecule comprising a single enantiomer. In someinstances, the polynucleic acid molecule comprises L-nucleotide. In someinstances, the polynucleic acid molecule comprises D-nucleotides. Insome instance, a polynucleic acid molecule composition comprises lessthan 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its mirrorenantiomer. In some cases, a polynucleic acid molecule compositioncomprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or lessof a racemic mixture. In some instances, the polynucleic acid moleculeis a polynucleic acid molecule described in: U.S. Patent PublicationNos: 2014/194610 and 2015/211006; and PCT Publication No.: WO2015107425.

In some embodiments, a polynucleic acid molecule described herein isfurther modified to include an aptamer conjugating moiety. In someinstances, the aptamer conjugating moiety is a DNA aptamer conjugatingmoiety. In some instances, the aptamer conjugating moiety is Alphamer(Centauri Therapeutics), which comprises an aptamer portion thatrecognizes a specific cell-surface target and a portion that presents aspecific epitopes for attaching to circulating antibodies. In someinstance, a polynucleic acid molecule described herein is furthermodified to include an aptamer conjugating moiety as described in: U.S.Pat. Nos. 8,604,184, 8,591,910, and 7,850,975.

In additional embodiments, a polynucleic acid molecule described hereinis modified to increase its stability. In some embodiment, thepolynucleic acid molecule is RNA (e.g., siRNA). In some instances, thepolynucleic acid molecule is modified by one or more of themodifications described above to increase its stability. In some cases,the polynucleic acid molecule is modified at the 2′ hydroxyl position,such as by 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl,2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA) modification or by a locked or bridgedribose conformation (e.g., LNA or ENA). In some cases, the polynucleicacid molecule is modified by 2′-O-methyl and/or 2′-O-methoxyethylribose. In some cases, the polynucleic acid molecule also includesmorpholinos, PNAs, HNA, methylphosphonate nucleotides, thiolphosphonatenucleotides, and/or 2′-fluoro N3-P5′-phosphoramidites to increase itsstability. In some instances, the polynucleic acid molecule is achirally pure (or stereo pure) polynucleic acid molecule. In someinstances, the chirally pure (or stereo pure) polynucleic acid moleculeis modified to increase its stability. Suitable modifications to the RNAto increase stability for delivery will be apparent to the skilledperson.

In some embodiments, a polynucleic acid molecule describe herein hasRNAi activity that modulates expression of RNA encoded by a geneinvolved in a disease or disorder such as, but not limited to, IKBKAP,SMN2, MCAD, LMNA, DMPK, ZNF9, MAPT, FKTN, TDP-43, LDLR, CFTR, DMD, PAH,MSTN, or K-Ras. In some instances, a polynucleic acid molecule describeherein is a double-stranded siRNA molecule that down-regulatesexpression of at least one of IKBKAP, SMN2, MCAD, LMNA, DMPK, ZNF9,MAPT, FKTN, TDP-43, LDLR, CFTR, DMD, PAH, MSTN, or K-Ras, wherein one ofthe strands of the double-stranded siRNA molecule comprises a nucleotidesequence that is complementary to a nucleotide sequence of at least oneof IKBKAP, SMN2, MCAD, LMNA, DMPK, ZNF9, MAPT, FKTN, TDP-43, LDLR, CFTR,DMD, PAH, MSTN, or K-Ras or RNA encoded by at least one of IKBKAP, SMN2,MCAD, LMNA, DMPK, ZNF9, MAPT, FKTN, TDP-43, LDLR, CFTR, DMD, PAH, MSTN,or K-Ras or a portion thereof, and wherein the second strand of thedouble-stranded siRNA molecule comprises a nucleotide sequencesubstantially similar to the nucleotide sequence of at least one ofIKBKAP, SMN2, MCAD, LMNA, DMPK, ZNF9, MAPT, FKTN, TDP-43, LDLR, CFTR,DMD, PAH, MSTN, or K-Ras or RNA encoded by at least one of IKBKAP, SMN2,MCAD, LMNA, DMPK, ZNF9, MAPT, FKTN, TDP-43, LDLR, CFTR, DMD, PAH, MSTN,or K-Ras or a portion thereof. In some cases, a polynucleic acidmolecule describe herein is a double-stranded siRNA molecule thatdown-regulates expression of at least one of IKBKAP, SMN2, MCAD, LMNA,DMPK, ZNF9, MAPT, FKTN, TDP-43, LDLR, CFTR, DMD, PAH, MSTN, or K-Ras,wherein each strand of the siRNA molecule comprises about 15 to 25, 18to 24, or 19 to about 23 nucleotides, and wherein each strand comprisesat least about 14, 17, or 19 nucleotides that are complementary to thenucleotides of the other strand. In some cases, a polynucleic acidmolecule describe herein is a double-stranded siRNA molecule thatdown-regulates expression of at least one of IKBKAP, SMN2, MCAD, LMNA,DMPK, ZNF9, MAPT, FKTN, TDP-43, LDLR, CFTR, DMD, PAH, MSTN, or K-Ras,wherein each strand of the siRNA molecule comprises about 19 to about 23nucleotides, and wherein each strand comprises at least about 19nucleotides that are complementary to the nucleotides of the otherstrand. In some instances, the RNAi activity occurs within a cell. Inother instances, the RNAi activity occurs in a reconstituted in vitrosystem.

In some embodiments, a polynucleic acid molecule describe herein hasRNAi activity that modulates expression of RNA encoded by a geneinvolved in muscular dystrophy such as, but not limited to, DMD, DUX4,DYSF, EMD, or LMNA. In some instances, a polynucleic acid moleculedescribe herein is a double-stranded siRNA molecule that down-regulatesexpression of at least one of DMD, DUX4, DYSF, EMD, or LMNA, wherein oneof the strands of the double-stranded siRNA molecule comprises anucleotide sequence that is complementary to a nucleotide sequence of atleast one of DMD, DUX4, DYSF, EMD, or LMINA or RNA encoded by at leastone of DMD, DUX4, DYSF, EMD, or LMINA or a portion thereof, and whereinthe second strand of the double-stranded siRNA molecule comprises anucleotide sequence substantially similar to the nucleotide sequence ofat least one of DMD, DUX4, DYSF, EMD, or LMINA or RNA encoded by atleast one of DMD, DUX4, DYSF, EMD, or LMINA or a portion thereof. Insome cases, a polynucleic acid molecule describe herein is adouble-stranded siRNA molecule that down-regulates expression of atleast one of DMD, DUX4, DYSF, EMD, or LMNA, wherein each strand of thesiRNA molecule comprises about 15 to 25, 18 to 24, or 19 to about 23nucleotides, and wherein each strand comprises at least about 14, 17, or19 nucleotides that are complementary to the nucleotides of the otherstrand. In some cases, a polynucleic acid molecule describe herein is adouble-stranded siRNA molecule that down-regulates expression of atleast one of DMD, DUX4, DYSF, EMD, or LMNA, wherein each strand of thesiRNA molecule comprises about 19 to about 23 nucleotides, and whereineach strand comprises at least about 19 nucleotides that arecomplementary to the nucleotides of the other strand. In some instances,the RNAi activity occurs within a cell. In other instances, the RNAiactivity occurs in a reconstituted in vitro system.

In some embodiments, a polynucleic acid molecule describe herein hasRNAi activity that modulates expression of RNA encoded by the DMD gene.In some instances, a polynucleic acid molecule describe herein is asingle-stranded siRNA molecule that down-regulates expression of DMD,wherein the single-stranded siRNA molecule comprises a nucleotidesequence that is complementary to a nucleotide sequence of DMD or RNAencoded by DMD or a portion thereof. In some cases, a polynucleic acidmolecule describe herein is a single-stranded siRNA molecule thatdown-regulates expression of DMD, wherein the siRNA molecule comprisesabout 15 to 25, 18 to 24, or 19 to about 23 nucleotides. In some cases,a polynucleic acid molecule describe herein is a single-stranded siRNAmolecule that down-regulates expression of DMD, wherein the siRNAmolecule comprises about 19 to about 23 nucleotides. In some instances,the RNAi activity occurs within a cell. In other instances, the RNAiactivity occurs in a reconstituted in vitro system.

In some instances, the polynucleic acid molecule is a double-strandedpolynucleotide molecule comprising self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a targetnucleic acid molecule or a portion thereof and the sense region havingnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof. In some instances, the polynucleic acid molecule isassembled from two separate polynucleotides, where one strand is thesense strand and the other is the antisense strand, wherein theantisense and sense strands are self-complementary (e.g., each strandcomprises nucleotide sequence that is complementary to nucleotidesequence in the other strand; such as where the antisense strand andsense strand form a duplex or double stranded structure, for examplewherein the double stranded region is about 19, 20, 21, 22, 23, or morebase pairs); the antisense strand comprises nucleotide sequence that iscomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof and the sense strand comprises nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof.Alternatively, the polynucleic acid molecule is assembled from a singleoligonucleotide, where the self-complementary sense and antisenseregions of the polynucleic acid molecule are linked by means of anucleic acid based or non-nucleic acid-based linker(s).

In some cases, the polynucleic acid molecule is a polynucleotide with aduplex, asymmetric duplex, hairpin or asymmetric hairpin secondarystructure, having self-complementary sense and antisense regions,wherein the antisense region comprises nucleotide sequence that iscomplementary to nucleotide sequence in a separate target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. In other cases, the polynucleic acid molecule is a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide is processed either in vivo or in vitro togenerate an active polynucleic acid molecule capable of mediating RNAi.In additional cases, the polynucleic acid molecule also comprises asingle stranded polynucleotide having nucleotide sequence complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof (for example, where such polynucleic acid molecule does notrequire the presence within the polynucleic acid molecule of nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof), wherein the single stranded polynucleotide further comprises aterminal phosphate group, such as a 5′-phosphate (see for exampleMartinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002,Molecular Cell, 10, 537-568), or 5′,3′-diphosphate.

In some instances, an asymmetric is a linear polynucleic acid moleculecomprising an antisense region, a loop portion that comprisesnucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complimentary nucleotides to base pair with theantisense region and form a duplex with loop. For example, an asymmetrichairpin polynucleic acid molecule comprises an antisense region havinglength sufficient to mediate RNAi in a cell or in vitro system (e.g.about 19 to about 22 nucleotides) and a loop region comprising about 4to about 8 nucleotides, and a sense region having about 3 to about 18nucleotides that are complementary to the antisense region. In somecases, the asymmetric hairpin polynucleic acid molecule also comprises a5′-terminal phosphate group that is chemically modified. In additionalcases, the loop portion of the asymmetric hairpin polynucleic acidmolecule comprises nucleotides, non-nucleotides, linker molecules, orconjugate molecules.

In some embodiments, an asymmetric duplex is a polynucleic acid moleculehaving two separate strands comprising a sense region and an antisenseregion, wherein the sense region comprises fewer nucleotides than theantisense region to the extent that the sense region has enoughcomplimentary nucleotides to base pair with the antisense region andform a duplex. For example, an asymmetric duplex polynucleic acidmolecule comprises an antisense region having length sufficient tomediate RNAi in a cell or in vitro system (e.g. about 19 to about 22nucleotides) and a sense region having about 3 to about 18 nucleotidesthat are complementary to the antisense region.

In some cases, an universal base refers to nucleotide base analogs thatform base pairs with each of the natural DNA/RNA bases with littlediscrimination between them. Non-limiting examples of universal basesinclude C-phenyl, C-naphthyl and other aromatic derivatives, inosine,azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole,4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (seefor example Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

Polynucleic Acid Molecule Synthesis

In some embodiments, a polynucleic acid molecule described herein isconstructed using chemical synthesis and/or enzymatic ligation reactionsusing procedures known in the art. For example, a polynucleic acidmolecule is chemically synthesized using naturally occurring nucleotidesor variously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the polynucleic acid molecule and target nucleicacids. Exemplary methods include those described in: U.S. Pat. Nos.5,142,047; 5,185,444; 5,889,136; 6,008,400; and 6,111,086; PCTPublication No. WO2009099942; or European Publication No. 1579015.Additional exemplary methods include those described in: Griffey et al.,“2′-O-aminopropyl ribonucleotides: a zwitterionic modification thatenhances the exonuclease resistance and biological activity of antisenseoligonucleotides,” J. Med. Chem. 39(26):5100-5109 (1997)); Obika, et al.“Synthesis of 2′-0,4′-C-methyleneuridine and -cytidine. Novel bicyclicnucleosides having a fixed C3, -endo sugar puckering”. TetrahedronLetters 38 (50): 8735 (1997); Koizumi, M. “ENA oligonucleotides astherapeutics”. Current opinion in molecular therapeutics 8 (2): 144-149(2006); and Abramova et al., “Novel oligonucleotide analogues based onmorpholino nucleoside subunits-antisense technologies: new chemicalpossibilities,” Indian Journal of Chemistry 48B:1721-1726 (2009).Alternatively, the polynucleic acid molecule is produced biologicallyusing an expression vector into which a polynucleic acid molecule hasbeen subcloned in an antisense orientation (i.e., RNA transcribed fromthe inserted polynucleic acid molecule will be of an antisenseorientation to a target polynucleic acid molecule of interest).

In some embodiments, a polynucleic acid molecule is synthesized via atandem synthesis methodology, wherein both strands are synthesized as asingle contiguous oligonucleotide fragment or strand separated by acleavable linker which is subsequently cleaved to provide separatefragments or strands that hybridize and permit purification of theduplex.

In some instances, a polynucleic acid molecule is also assembled fromtwo distinct nucleic acid strands or fragments wherein one fragmentincludes the sense region and the second fragment includes the antisenseregion of the molecule.

Additional modification methods for incorporating, for example, sugar,base and phosphate modifications include: Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344,565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren,Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. InternationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al.,International PCT publication No. WO 97/26270; Beigelman et al., U.S.Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,International PCT Publication No. WO 98/13526; Thompson et al., U.S.Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al.,1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers(Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev.Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5,1999-2010. Such publications describe general methods and strategies todetermine the location of incorporation of sugar, base and/or phosphatemodifications and the like into nucleic acid molecules withoutmodulating catalysis.

In some instances, while chemical modification of the polynucleic acidmolecule internucleotide linkages with phosphorothioate,phosphorodithioate, and/or 5′-methylphosphonate linkages improvesstability, excessive modifications sometimes cause toxicity or decreasedactivity. Therefore, when designing nucleic acid molecules, the amountof these internucleotide linkages in some cases is minimized. In suchcases, the reduction in the concentration of these linkages lowerstoxicity, increases efficacy and higher specificity of these molecules.

Nucleic Acid-Polypeptide Conjugate

In some embodiments, a polynucleic acid molecule is further conjugatedto a polypeptide A for delivery to a site of interest. In some cases, apolynucleic acid molecule is conjugated to a polypeptide A andoptionally a polymeric moiety.

In some instances, at least one polypeptide A is conjugated to at leastone B. In some instances, the at least one polypeptide A is conjugatedto the at least one B to form an A-B conjugate. In some embodiments, atleast one A is conjugated to the 5′ terminus of B, the 3′ terminus of B,an internal site on B, or in any combinations thereof. In someinstances, the at least one polypeptide A is conjugated to at least twoB. In some instances, the at least one polypeptide A is conjugated to atleast 2, 3, 4, 5, 6, 7, 8, or more B.

In some embodiments, at least one polypeptide A is conjugated at oneterminus of at least one B while at least one C is conjugated at theopposite terminus of the at least one B to form an A-B—C conjugate. Insome instances, at least one polypeptide A is conjugated at one terminusof the at least one B while at least one of C is conjugated at aninternal site on the at least one B. In some instances, at least onepolypeptide A is conjugated directly to the at least one C. In someinstances, the at least one B is conjugated indirectly to the at leastone polypeptide A via the at least one C to form an A-C—B conjugate.

In some instances, at least one B and/or at least one C, and optionallyat least one D are conjugated to at least one polypeptide A. In someinstances, the at least one B is conjugated at a terminus (e.g., a 5′terminus or a 3′ terminus) to the at least one polypeptide A or areconjugated via an internal site to the at least one polypeptide A. Insome cases, the at least one C is conjugated either directly to the atleast one polypeptide A or indirectly via the at least one B. Ifindirectly via the at least one B, the at least one C is conjugatedeither at the same terminus as the at least one polypeptide A on B, atopposing terminus from the at least one polypeptide A, or independentlyat an internal site. In some instances, at least one additionalpolypeptide A is further conjugated to the at least one polypeptide A,to B, or to C. In additional instances, the at least one D is optionallyconjugated either directly or indirectly to the at least one polypeptideA, to the at least one B, or to the at least one C. If directly to theat least one polypeptide A, the at least one D is also optionallyconjugated to the at least one B to form an A-D-B conjugate or isoptionally conjugated to the at least one B and the at least one C toform an A-D-B—C conjugate. In some instances, the at least one D isdirectly conjugated to the at least one polypeptide A and indirectly tothe at least one B and the at least one C to form a D-A-B—C conjugate.If indirectly to the at least one polypeptide A, the at least one D isalso optionally conjugated to the at least one B to form an A-B-Dconjugate or is optionally conjugated to the at least one B and the atleast one C to form an A-B-D-C conjugate. In some instances, at leastone additional D is further conjugated to the at least one polypeptideA, to B, or to C.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19A.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19B.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19C.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19D.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19E.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19F.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19G.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19H.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19I.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19J.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19K.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19L.

The antibody as illustrated above is for representation purposes onlyand encompasses a humanized antibody or binding fragment thereof,chimeric antibody or binding fragment thereof, monoclonal antibody orbinding fragment thereof, monovalent Fab′, divalent Fab2, single-chainvariable fragment (scFv), diabody, minibody, nanobody, single-domainantibody (sdAb), or camelid antibody or binding fragment thereof.

Binding Moiety

In some embodiments, the binding moiety A is a polypeptide. In someinstances, the polypeptide is an antibody or its fragment thereof. Insome cases, the fragment is a binding fragment. In some instances, theantibody or binding fragment thereof comprises a humanized antibody orbinding fragment thereof, murine antibody or binding fragment thereof,chimeric antibody or binding fragment thereof, monoclonal antibody orbinding fragment thereof, monovalent Fab′, divalent Fab₂, F(ab)′₃fragments, single-chain variable fragment (scFv), bis-scFv, (scFv)₂,diabody, minibody, nanobody, triabody, tetrabody, disulfide stabilizedFv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelidantibody or binding fragment thereof, bispecific antibody or bidingfragment thereof, or a chemically modified derivative thereof.

In some instances, A is an antibody or binding fragment thereof. In someinstances, A is a humanized antibody or binding fragment thereof, murineantibody or binding fragment thereof, chimeric antibody or bindingfragment thereof, monoclonal antibody or binding fragment thereof,monovalent Fab′, divalent Fab₂, F(ab)′₃ fragments, single-chain variablefragment (scFv), bis-scFv, (scFv)₂, diabody, minibody, nanobody,triabody, tetrabody, disulfide stabilized Fv protein (“dsFv”),single-domain antibody (sdAb), Ig NAR, camelid antibody or bindingfragment thereof, bispecific antibody or biding fragment thereof, or achemically modified derivative thereof. In some instances, A is ahumanized antibody or binding fragment thereof. In some instances, A isa murine antibody or binding fragment thereof. In some instances, A is achimeric antibody or binding fragment thereof. In some instances, A is amonoclonal antibody or binding fragment thereof. In some instances, A isa monovalent Fab′. In some instances, A is a diavalent Fab₂. In someinstances, A is a single-chain variable fragment (scFv).

In some embodiments, the binding moiety A is a bispecific antibody orbinding fragment thereof. In some instances, the bispecific antibody isa trifunctional antibody or a bispecific mini-antibody. In some cases,the bispecific antibody is a trifunctional antibody. In some instances,the trifunctional antibody is a full length monoclonal antibodycomprising binding sites for two different antigens.

In some cases, the bispecific antibody is a bispecific mini-antibody. Insome instances, the bispecific mini-antibody comprises divalent Fab₂,F(ab)′₃ fragments, bis-scFv, (scFv)₂, diabody, minibody, triabody,tetrabody or a bi-specific T-cell engager (BiTE). In some embodiments,the bi-specific T-cell engager is a fusion protein that contains twosingle-chain variable fragments (scFvs) in which the two scFvs targetepitopes of two different antigens.

In some embodiments, the binding moiety A is a bispecific mini-antibody.In some instances, A is a bispecific Fab₂. In some instances, A is abispecific F(ab)′₃ fragment. In some cases, A is a bispecific bis-scFv.In some cases, A is a bispecific (scFv)₂. In some embodiments, A is abispecific diabody. In some embodiments, A is a bispecific minibody. Insome embodiments, A is a bispecific triabody. In other embodiments, A isa bispecific tetrabody. In other embodiments, A is a bi-specific T-cellengager (BiTE).

In some embodiments, the binding moiety A is a trispecific antibody. Insome instances, the trispecific antibody comprises F(ab)′₃ fragments ora triabody. In some instances, A is a trispecific F(ab)′₃ fragment. Insome cases, A is a triabody. In some embodiments, A is a trispecificantibody as described in Dimas, et al., “Development of a trispecificantibody designed to simultaneously and efficiently target threedifferent antigens on tumor cells,” Mol. Pharmaceutics, 12(9): 3490-3501(2015).

In some embodiments, the binding moiety A is an antibody or bindingfragment thereof that recognizes a cell surface protein. In someinstances, the binding moiety A is an antibody or binding fragmentthereof that recognizes a cell surface protein on a muscle cell.Exemplary cell surface proteins recognized by an antibody or bindingfragment thereof include, but are not limited to, Sca-1, CD34, Myo-D,myogenin, MRF4, NCAM, CD43, and CD95 (Fas).

In some instances, the cell surface protein comprises clusters ofdifferentiation (CD) cell surface markers. Exemplary CD cell surfacemarkers include, but are not limited to, CD1, CD2, CD3, CD4, CD5, CD6,CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14,CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24,CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36,CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD45RO, CD45RA,CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f,CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61,CD62E, CD62L (L-selectin), CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c,CD66d, CD66e, CD79 (e.g., CD79a, CD79b), CD90, CD95 (Fas), CD103, CD104,CD125 (IL5RA), CD134 (OX40), CD137 (4-1BB), CD152 (CTLA-4), CD221,CD274, CD279 (PD-1), CD319 (SLAMF7), CD326 (EpCAM), and the like.

In some instances, the binding moiety A is an antibody or bindingfragment thereof that recognizes a CD cell surface marker. In someinstances, the binding moiety A is an antibody or binding fragmentthereof that recognizes CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9,CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD15s, CD16,CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28,CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40,CD41, CD42, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48,CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54,CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L (L-selectin),CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD79 (e.g.,CD79a, CD79b), CD90, CD95 (Fas), CD103, CD104, CD125 (IL5RA), CD134(OX40), CD137 (4-1BB), CD152 (CTLA-4), CD221, CD274, CD279 (PD-1), CD319(SLAMF7), CD326 (EpCAM), or a combination thereof.

In some embodiments, the binding moiety A is conjugated to a polynucleicacid molecule (B) non-specifically. In some instances, the bindingmoiety A is conjugated to a polynucleic acid molecule (B) via a lysineresidue or a cysteine residue, in a non-site specific manner. In someinstances, the binding moiety A is conjugated to a polynucleic acidmolecule (B) via a lysine residue in a non-site specific manner. In somecases, the binding moiety A is conjugated to a polynucleic acid molecule(B) via a cysteine residue in a non-site specific manner.

In some embodiments, the binding moiety A is conjugated to a polynucleicacid molecule (B) in a site-specific manner. In some instances, thebinding moiety A is conjugated to a polynucleic acid molecule (B)through a lysine residue, a cysteine residue, at the 5′-terminus, at the3′-terminus, an unnatural amino acid, or an enzyme-modified orenzyme-catalyzed residue, via a site-specific manner. In some instances,the binding moiety A is conjugated to a polynucleic acid molecule (B)through a lysine residue via a site-specific manner. In some instances,the binding moiety A is conjugated to a polynucleic acid molecule (B)through a cysteine residue via a site-specific manner. In someinstances, the binding moiety A is conjugated to a polynucleic acidmolecule (B) at the 5′-terminus via a site-specific manner. In someinstances, the binding moiety A is conjugated to a polynucleic acidmolecule (B) at the 3′-terminus via a site-specific manner. In someinstances, the binding moiety A is conjugated to a polynucleic acidmolecule (B) through an unnatural amino acid via a site-specific manner.In some instances, the binding moiety A is conjugated to a polynucleicacid molecule (B) through an enzyme-modified or enzyme-catalyzed residuevia a site-specific manner.

In some embodiments, one or more polynucleic acid molecule (B) isconjugated to a binding moiety A. In some instances, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 1 polynucleic acid molecule is conjugated to one binding moiety A.In some instances, about 2 polynucleic acid molecules are conjugated toone binding moiety A. In some instances, about 3 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 4 polynucleic acid molecules are conjugated to one binding moietyA. In some instances, about 5 polynucleic acid molecules are conjugatedto one binding moiety A. In some instances, about 6 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 7 polynucleic acid molecules are conjugated to one binding moietyA. In some instances, about 8 polynucleic acid molecules are conjugatedto one binding moiety A. In some instances, about 9 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 10 polynucleic acid molecules are conjugated to one binding moietyA. In some instances, about 11 polynucleic acid molecules are conjugatedto one binding moiety A. In some instances, about 12 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 13 polynucleic acid molecules are conjugated to one binding moietyA. In some instances, about 14 polynucleic acid molecules are conjugatedto one binding moiety A. In some instances, about 15 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 16 polynucleic acid molecules are conjugated to one binding moietyA. In some cases, the one or more polynucleic acid molecules are thesame. In other cases, the one or more polynucleic acid molecules aredifferent.

In some embodiments, the number of polynucleic acid molecule (B)conjugated to a binding moiety A forms a ratio. In some instances, theratio is referred to as a DAR (drug-to-antibody) ratio, in which thedrug as referred to herein is the polynucleic acid molecule (B). In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,or greater. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is about 1 or greater. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 2 or greater. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 3 or greater.In some instances, the DAR ratio of the polynucleic acid molecule (B) tobinding moiety A is about 4 or greater. In some instances, the DAR ratioof the polynucleic acid molecule (B) to binding moiety A is about 5 orgreater. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is about 6 or greater. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 7 or greater. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 8 or greater.In some instances, the DAR ratio of the polynucleic acid molecule (B) tobinding moiety A is about 9 or greater. In some instances, the DAR ratioof the polynucleic acid molecule (B) to binding moiety A is about 10 orgreater. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is about 11 or greater. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 12 or greater.

In some instances, the DAR ratio of the polynucleic acid molecule (B) tobinding moiety A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, or 16. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is about 1. In some instances, the DARratio of the polynucleic acid molecule (B) to binding moiety A is about2. In some instances, the DAR ratio of the polynucleic acid molecule (B)to binding moiety A is about 3. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 4. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 5. In some instances, the DAR ratio of the polynucleicacid molecule (B) to binding moiety A is about 6. In some instances, theDAR ratio of the polynucleic acid molecule (B) to binding moiety A isabout 7. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is about 8. In some instances, the DARratio of the polynucleic acid molecule (B) to binding moiety A is about9. In some instances, the DAR ratio of the polynucleic acid molecule (B)to binding moiety A is about 10. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 11. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 12. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 13. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 14. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 15. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 16.

In some instances, the DAR ratio of the polynucleic acid molecule (B) tobinding moiety A is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,or 16. In some instances, the DAR ratio of the polynucleic acid molecule(B) to binding moiety A is 1. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is 2. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is 4. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is 6. In some instances, the DAR ratioof the polynucleic acid molecule (B) to binding moiety A is 8. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is 12.

In some instances, a conjugate comprising polynucleic acid molecule (B)and binding moiety A has improved activity as compared to a conjugatecomprising polynucleic acid molecule (B) without a binding moiety A. Insome instances, improved activity results in enhanced biologicallyrelevant functions, e.g., improved stability, affinity, binding,functional activity, and efficacy in treatment or prevention of adisease state. In some instances, the disease state is a result of oneor more mutated exons of a gene. In some instances, the conjugatecomprising polynucleic acid molecule (B) and binding moiety A results inincreased exon skipping of the one or more mutated exons as compared tothe conjugate comprising polynucleic acid molecule (B) without a bindingmoiety A. In some instances, exon skipping is increased by at least orabout 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or morethan 95% in the conjugate comprising polynucleic acid molecule (B) andbinding moiety A as compared to the conjugate comprising polynucleicacid molecule (B) without a binding moiety A.

In some embodiments, an antibody or its binding fragment is furthermodified using conventional techniques known in the art, for example, byusing amino acid deletion, insertion, substitution, addition, and/or byrecombination and/or any other modification (e.g. posttranslational andchemical modifications, such as glycosylation and phosphorylation) knownin the art either alone or in combination. In some instances, themodification further comprises a modification for modulating interactionwith Fc receptors. In some instances, the one or more modificationsinclude those described in, for example, International Publication No.WO97/34631, which discloses amino acid residues involved in theinteraction between the Fc domain and the FcRn receptor. Methods forintroducing such modifications in the nucleic acid sequence underlyingthe amino acid sequence of an antibody or its binding fragment is wellknown to the person skilled in the art.

In some instances, an antibody binding fragment further encompasses itsderivatives and includes polypeptide sequences containing at least oneCDR.

In some instances, the term “single-chain” as used herein means that thefirst and second domains of a bi-specific single chain construct arecovalently linked, preferably in the form of a co-linear amino acidsequence encodable by a single nucleic acid molecule.

In some instances, a bispecific single chain antibody construct relatesto a construct comprising two antibody derived binding domains. In suchembodiments, bi-specific single chain antibody construct is tandembi-scFv or diabody. In some instances, a scFv contains a VH and VLdomain connected by a linker peptide. In some instances, linkers are ofa length and sequence sufficient to ensure that each of the first andsecond domains can, independently from one another, retain theirdifferential binding specificities.

In some embodiments, binding to or interacting with as used hereindefines a binding/interaction of at least two antigen-interaction-siteswith each other. In some instances, antigen-interaction-site defines amotif of a polypeptide that shows the capacity of specific interactionwith a specific antigen or a specific group of antigens. In some cases,the binding/interaction is also understood to define a specificrecognition. In such cases, specific recognition refers to that theantibody or its binding fragment is capable of specifically interactingwith and/or binding to at least two amino acids of each of a targetmolecule. For example, specific recognition relates to the specificityof the antibody molecule, or to its ability to discriminate between thespecific regions of a target molecule. In additional instances, thespecific interaction of the antigen-interaction-site with its specificantigen results in an initiation of a signal, e.g. due to the inductionof a change of the conformation of the antigen, an oligomerization ofthe antigen, etc. In further embodiments, the binding is exemplified bythe specificity of a “key-lock-principle”. Thus in some instances,specific motifs in the amino acid sequence of theantigen-interaction-site and the antigen bind to each other as a resultof their primary, secondary or tertiary structure as well as the resultof secondary modifications of said structure. In such cases, thespecific interaction of the antigen-interaction-site with its specificantigen results as well in a simple binding of the site to the antigen.

In some instances, specific interaction further refers to a reducedcross-reactivity of the antibody or its binding fragment or a reducedoff-target effect. For example, the antibody or its binding fragmentthat bind to the polypeptide/protein of interest but do not or do notessentially bind to any of the other polypeptides are considered asspecific for the polypeptide/protein of interest. Examples for thespecific interaction of an antigen-interaction-site with a specificantigen comprise the specificity of a ligand for its receptor, forexample, the interaction of an antigenic determinant (epitope) with theantigenic binding site of an antibody.

Conjugation Chemistry

In some embodiments, a polynucleic acid molecule B is conjugated to abinding moiety. In some instances, the binding moiety comprises aminoacids, peptides, polypeptides, proteins, antibodies, antigens, toxins,hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates,polymers such as polyethylene glycol and polypropylene glycol, as wellas analogs or derivatives of all of these classes of substances.Additional examples of binding moiety also include steroids, such ascholesterol, phospholipids, di- and triacylglycerols, fatty acids,hydrocarbons (e.g., saturated, unsaturated, or contains substitutions),enzyme substrates, biotin, digoxigenin, and polysaccharides. In someinstances, the binding moiety is an antibody or binding fragmentthereof. In some instances, the polynucleic acid molecule is furtherconjugated to a polymer, and optionally an endosomolytic moiety.

In some embodiments, the polynucleic acid molecule is conjugated to thebinding moiety by a chemical ligation process. In some instances, thepolynucleic acid molecule is conjugated to the binding moiety by anative ligation. In some instances, the conjugation is as described in:Dawson, et al. “Synthesis of proteins by native chemical ligation,”Science 1994, 266, 776-779; Dawson, et al. “Modulation of Reactivity inNative Chemical Ligation through the Use of Thiol Additives,” J. Am.Chem. Soc. 1997, 119, 4325-4329; Hackeng, et al. “Protein synthesis bynative chemical ligation: Expanded scope by using straightforwardmethodology.,” Proc. Natl. Acad. Sci. USA 1999, 96, 10068-10073; or Wu,et al. “Building complex glycopeptides: Development of a cysteine-freenative chemical ligation protocol,” Angew. Chem. Int. Ed. 2006, 45,4116-4125. In some instances, the conjugation is as described in U.S.Pat. No. 8,936,910. In some embodiments, the polynucleic acid moleculeis conjugated to the binding moiety either site-specifically ornon-specifically via native ligation chemistry.

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a site-directed method utilizing a “traceless”coupling technology (Philochem). In some instances, the “traceless”coupling technology utilizes an N-terminal 1,2-aminothiol group on thebinding moiety which is then conjugate with a polynucleic acid moleculecontaining an aldehyde group. (see Casi et al., “Site-specific tracelesscoupling of potent cytotoxic drugs to recombinant antibodies forpharmacodelivery,” JACS 134(13): 5887-5892 (2012))

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a site-directed method utilizing an unnatural aminoacid incorporated into the binding moiety. In some instances, theunnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In someinstances, the keto group of pAcPhe is selectively coupled to analkoxy-amine derivatived conjugating moiety to form an oxime bond. (seeAxup et al., “Synthesis of site-specific antibody-drug conjugates usingunnatural amino acids,” PNAS 109(40): 16101-16106 (2012)).

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a site-directed method utilizing an enzyme-catalyzedprocess. In some instances, the site-directed method utilizes SMARTag™technology (Redwood). In some instances, the SMARTag™ technologycomprises generation of a formylglycine (FGly) residue from cysteine byformylglycine-generating enzyme (FGE) through an oxidation process underthe presence of an aldehyde tag and the subsequent conjugation of FGlyto an alkylhydraine-functionalized polynucleic acid molecule viahydrazino-Pictet-Spengler (HIPS) ligation. (see Wu et al.,“Site-specific chemical modification of recombinant proteins produced inmammalian cells by using the genetically encoded aldehyde tag,” PNAS106(9): 3000-3005 (2009); Agarwal, et al., “A Pictet-Spengler ligationfor protein chemical modification,” PNAS 110(1): 46-51 (2013))

In some instances, the enzyme-catalyzed process comprises microbialtransglutaminase (mTG). In some cases, the polynucleic acid molecule isconjugated to the binding moiety utilizing a microbial transglutaminzecatalyzed process. In some instances, mTG catalyzes the formation of acovalent bond between the amide side chain of a glutamine within therecognition sequence and a primary amine of a functionalized polynucleicacid molecule. In some instances, mTG is produced from Streptomycesmobarensis. (see Strop et al., “Location matters: site of conjugationmodulates stability and pharmacokinetics of antibody drug conjugates,”Chemistry and Biology 20(2) 161-167 (2013))

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a method as described in PCT Publication No.WO2014/140317, which utilizes a sequence-specific transpeptidase.

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a method as described in U.S. Patent Publication Nos.2015/0105539 and 2015/0105540.

Production of Antibodies or Binding Fragments Thereof

In some embodiments, polypeptides described herein (e.g., antibodies andits binding fragments) are produced using any method known in the art tobe useful for the synthesis of polypeptides (e.g., antibodies), inparticular, by chemical synthesis or by recombinant expression, and arepreferably produced by recombinant expression techniques.

In some instances, an antibody or its binding fragment thereof isexpressed recombinantly, and the nucleic acid encoding the antibody orits binding fragment is assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., 1994,BioTechniques 17:242), which involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligation of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a nucleic acid molecule encoding an antibody isoptionally generated from a suitable source (e.g., an antibody cDNAlibrary, or cDNA library generated from any tissue or cells expressingthe immunoglobulin) by PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence.

In some instances, an antibody or its binding is optionally generated byimmunizing an animal, such as a rabbit, to generate polyclonalantibodies or, more preferably, by generating monoclonal antibodies,e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or,as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole etal. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96). Alternatively, a clone encoding at least the Fabportion of the antibody is optionally obtained by screening Fabexpression libraries (e.g., as described in Huse et al., 1989, Science246:1275-1281) for clones of Fab fragments that bind the specificantigen or by screening antibody libraries (See, e.g., Clackson et al.,1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA94:4937).

In some embodiments, techniques developed for the production of“chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al.,1985, Nature 314:452-454) by splicing genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity are used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region, e.g., humanized antibodies.

In some embodiments, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are adapted toproduce single chain antibodies. Single chain antibodies are formed bylinking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide. Techniquesfor the assembly of functional Fv fragments in E. coli are alsooptionally used (Skerra et al., 1988, Science 242:1038-1041).

In some embodiments, an expression vector comprising the nucleotidesequence of an antibody or the nucleotide sequence of an antibody istransferred to a host cell by conventional techniques (e.g.,electroporation, liposomal transfection, and calcium phosphateprecipitation), and the transfected cells are then cultured byconventional techniques to produce the antibody. In specificembodiments, the expression of the antibody is regulated by aconstitutive, an inducible or a tissue, specific promoter.

In some embodiments, a variety of host-expression vector systems isutilized to express an antibody or its binding fragment describedherein. Such host-expression systems represent vehicles by which thecoding sequences of the antibody is produced and subsequently purified,but also represent cells that are, when transformed or transfected withthe appropriate nucleotide coding sequences, express an antibody or itsbinding fragment in situ. These include, but are not limited to,microorganisms such as bacteria (e.g., E. coli and B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing an antibody or its binding fragmentcoding sequences; yeast (e.g., Saccharomyces Pichia) transformed withrecombinant yeast expression vectors containing an antibody or itsbinding fragment coding sequences; insect cell systems infected withrecombinant virus expression vectors (e.g., baculovirus) containing anantibody or its binding fragment coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containing anantibody or its binding fragment coding sequences; or mammalian cellsystems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5Kpromoter).

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. In some instances, cell lines that stablyexpress an antibody are optionally engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellsare transformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells are thenallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci that in turnare cloned and expanded into cell lines. This method can advantageouslybe used to engineer cell lines which express the antibody or its bindingfragments.

In some instances, a number of selection systems are used, including butnot limited to the herpes simplex virus thymidine kinase (Wigler et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), andadenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genesare employed in tk−, hgprt− or aprt− cells, respectively. Also,antimetabolite resistance are used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan,1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev.Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215) and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds., 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY;and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, CurrentProtocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin etal., 1981, J. Mol. Biol. 150:1).

In some instances, the expression levels of an antibody are increased byvector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing an antibodyis amplifiable, an increase in the level of inhibitor present in cultureof host cell will increase the number of copies of the marker gene.Since the amplified region is associated with the nucleotide sequence ofthe antibody, production of the antibody will also increase (Crouse etal., 1983, Mol. Cell Biol. 3:257).

In some instances, any method known in the art for purification oranalysis of an antibody or antibody conjugates is used, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Exemplarychromatography methods included, but are not limited to, strong anionexchange chromatography, hydrophobic interaction chromatography, sizeexclusion chromatography, and fast protein liquid chromatography.

Polymer Conjugating Moiety

In some embodiments, a polymer moiety C is further conjugated to apolynucleic acid molecule described herein, a binding moiety describedherein, or in combinations thereof. In some instances, a polymer moietyC is conjugated a polynucleic acid molecule. In some cases, a polymermoiety C is conjugated to a binding moiety. In other cases, a polymermoiety C is conjugated to a polynucleic acid molecule-binding moietymolecule. In additional cases, a polymer moiety C is conjugated, asillustrated supra.

In some instances, the polymer moiety C is a natural or syntheticpolymer, consisting of long chains of branched or unbranched monomers,and/or cross-linked network of monomers in two or three dimensions. Insome instances, the polymer moiety C includes a polysaccharide, lignin,rubber, or polyalkylen oxide (e.g., polyethylene glycol). In someinstances, the at least one polymer moiety C includes, but is notlimited to, alpha-, omega-dihydroxylpolyethyleneglycol, biodegradablelactone-based polymer, e.g. polyacrylic acid, polylactide acid (PLA),poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefin,polyamide, polycyanoacrylate, polyimide, polyethylenterephthalat (PET,PETG), polyethylene terephthalate (PETE), polytetramethylene glycol(PTG), or polyurethane as well as mixtures thereof. As used herein, amixture refers to the use of different polymers within the same compoundas well as in reference to block copolymers. In some cases, blockcopolymers are polymers wherein at least one section of a polymer isbuild up from monomers of another polymer. In some instances, thepolymer moiety C comprises polyalkylene oxide. In some instances, thepolymer moiety C comprises PEG. In some instances, the polymer moiety Ccomprises polyethylene imide (PEI) or hydroxy ethyl starch (HES).

In some instances, C is a PEG moiety. In some instances, the PEG moietyis conjugated at the 5′ terminus of the polynucleic acid molecule whilethe binding moiety is conjugated at the 3′ terminus of the polynucleicacid molecule. In some instances, the PEG moiety is conjugated at the 3′terminus of the polynucleic acid molecule while the binding moiety isconjugated at the 5′ terminus of the polynucleic acid molecule. In someinstances, the PEG moiety is conjugated to an internal site of thepolynucleic acid molecule. In some instances, the PEG moiety, thebinding moiety, or a combination thereof, are conjugated to an internalsite of the polynucleic acid molecule. In some instances, theconjugation is a direct conjugation. In some instances, the conjugationis via native ligation.

In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydispersor monodispers compound. In some instances, polydispers materialcomprises disperse distribution of different molecular weight of thematerial, characterized by mean weight (weight average) size anddispersity. In some instances, the monodisperse PEG comprises one sizeof molecules. In some embodiments, C is poly- or monodispersedpolyalkylene oxide (e.g., PEG) and the indicated molecular weightrepresents an average of the molecular weight of the polyalkylene oxide,e.g., PEG, molecules.

In some embodiments, the molecular weight of the polyalkylene oxide(e.g., PEG) is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100,1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750,4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000,10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.

In some embodiments, C is polyalkylene oxide (e.g., PEG) and has amolecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100,2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500,3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500,8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000Da. In some embodiments, C is PEG and has a molecular weight of about200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500,2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500,4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000,20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In someinstances, the molecular weight of C is about 200 Da. In some instances,the molecular weight of C is about 300 Da. In some instances, themolecular weight of C is about 400 Da. In some instances, the molecularweight of C is about 500 Da. In some instances, the molecular weight ofC is about 600 Da. In some instances, the molecular weight of C is about700 Da. In some instances, the molecular weight of C is about 800 Da. Insome instances, the molecular weight of C is about 900 Da. In someinstances, the molecular weight of C is about 1000 Da. In someinstances, the molecular weight of C is about 1100 Da. In someinstances, the molecular weight of C is about 1200 Da. In someinstances, the molecular weight of C is about 1300 Da. In someinstances, the molecular weight of C is about 1400 Da. In someinstances, the molecular weight of C is about 1450 Da. In someinstances, the molecular weight of C is about 1500 Da. In someinstances, the molecular weight of C is about 1600 Da. In someinstances, the molecular weight of C is about 1700 Da. In someinstances, the molecular weight of C is about 1800 Da. In someinstances, the molecular weight of C is about 1900 Da. In someinstances, the molecular weight of C is about 2000 Da. In someinstances, the molecular weight of C is about 2100 Da. In someinstances, the molecular weight of C is about 2200 Da. In someinstances, the molecular weight of C is about 2300 Da. In someinstances, the molecular weight of C is about 2400 Da. In someinstances, the molecular weight of C is about 2500 Da. In someinstances, the molecular weight of C is about 2600 Da. In someinstances, the molecular weight of C is about 2700 Da. In someinstances, the molecular weight of C is about 2800 Da. In someinstances, the molecular weight of C is about 2900 Da. In someinstances, the molecular weight of C is about 3000 Da. In someinstances, the molecular weight of C is about 3250 Da. In someinstances, the molecular weight of C is about 3350 Da. In someinstances, the molecular weight of C is about 3500 Da. In someinstances, the molecular weight of C is about 3750 Da. In someinstances, the molecular weight of C is about 4000 Da. In someinstances, the molecular weight of C is about 4250 Da. In someinstances, the molecular weight of C is about 4500 Da. In someinstances, the molecular weight of C is about 4600 Da. In someinstances, the molecular weight of C is about 4750 Da. In someinstances, the molecular weight of C is about 5000 Da. In someinstances, the molecular weight of C is about 5500 Da. In someinstances, the molecular weight of C is about 6000 Da. In someinstances, the molecular weight of C is about 6500 Da. In someinstances, the molecular weight of C is about 7000 Da. In someinstances, the molecular weight of C is about 7500 Da. In someinstances, the molecular weight of C is about 8000 Da. In someinstances, the molecular weight of C is about 10,000 Da. In someinstances, the molecular weight of C is about 12,000 Da. In someinstances, the molecular weight of C is about 20,000 Da. In someinstances, the molecular weight of C is about 35,000 Da. In someinstances, the molecular weight of C is about 40,000 Da. In someinstances, the molecular weight of C is about 50,000 Da. In someinstances, the molecular weight of C is about 60,000 Da. In someinstances, the molecular weight of C is about 100,000 Da.

In some embodiments, the polyalkylene oxide (e.g., PEG) comprisesdiscrete ethylene oxide units (e.g., four to about 48 ethylene oxideunits). In some instances, the polyalkylene oxide comprising thediscrete ethylene oxide units is a linear chain. In other cases, thepolyalkylene oxide comprising the discrete ethylene oxide units is abranched chain.

In some instances, the polymer moiety C is a polyalkylene oxide (e.g.,PEG) comprising discrete ethylene oxide units. In some cases, thepolymer moiety C comprises between about 4 and about 48 ethylene oxideunits. In some cases, the polymer moiety C comprises about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14, about 15, about 16, about 17, about 18, about 19, about20, about 21, about 22, about 23, about 24, about 25, about 26, about27, about 28, about 29, about 30, about 31, about 32, about 33, about34, about 35, about 36, about 37, about 38, about 39, about 40, about41, about 42, about 43, about 44, about 45, about 46, about 47, or about48 ethylene oxide units.

In some instances, the polymer moiety C is a discrete PEG comprising,e.g., between about 4 and about 48 ethylene oxide units. In some cases,the polymer moiety C is a discrete PEG comprising, e.g., about 4, about5, about 6, about 7, about 8, about 9, about 10, about 11, about 12,about 13, about 14, about 15, about 16, about 17, about 18, about 19,about 20, about 21, about 22, about 23, about 24, about 25, about 26,about 27, about 28, about 29, about 30, about 31, about 32, about 33,about 34, about 35, about 36, about 37, about 38, about 39, about 40,about 41, about 42, about 43, about 44, about 45, about 46, about 47, orabout 48 ethylene oxide units. In some cases, the polymer moiety C is adiscrete PEG comprising, e.g., about 4 ethylene oxide units. In somecases, the polymer moiety C is a discrete PEG comprising, e.g., about 5ethylene oxide units. In some cases, the polymer moiety C is a discretePEG comprising, e.g., about 6 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 7 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 8 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 9 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 10 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 11 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 12 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 13 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 14 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 15 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 16 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 17 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 18 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 19 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 20 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 21 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 22 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 23 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 24 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 25 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 26 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 27 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 28 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 29 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 30 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 31 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 32 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 33 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 34 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 35 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 36 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 37 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 38 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 39 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 40 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 41 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 42 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 43 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 44 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 45 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 46 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 47 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 48 ethylene oxide units.

In some cases, the polymer moiety C is dPEG® (Quanta Biodesign Ltd).

In some embodiments, the polymer moiety C comprises a cationic mucicacid-based polymer (cMAP). In some instances, cMAP comprises one or moresubunit of at least one repeating subunit, and the subunit structure isrepresented as Formula (V):

wherein m is independently at each occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10, preferably 4-6 or 5; and n is independently at each occurrence 1,2, 3, 4, or 5. In some embodiments, m and n are, for example, about 10.

In some instances, cMAP is further conjugated to a PEG moiety,generating a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, ora cMAP-PEG-cMAP triblock polymer. In some instances, the PEG moiety isin a range of from about 500 Da to about 50,000 Da. In some instances,the PEG moiety is in a range of from about 500 Da to about 1000 Da,greater than 1000 Da to about 5000 Da, greater than 5000 Da to about10,000 Da, greater than 10,000 to about 25,000 Da, greater than 25,000Da to about 50,000 Da, or any combination of two or more of theseranges.

In some instances, the polymer moiety C is cMAP-PEG copolymer, anmPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. Insome cases, the polymer moiety C is cMAP-PEG copolymer. In other cases,the polymer moiety C is an mPEG-cMAP-PEGm triblock polymer. Inadditional cases, the polymer moiety C is a cMAP-PEG-cMAP triblockpolymer.

In some embodiments, the polymer moiety C is conjugated to thepolynucleic acid molecule, the binding moiety, and optionally to theendosomolytic moiety as illustrated supra.

Endosomolytic Moiety

In some embodiments, a molecule of Formula (I): A-X—B—Y—C, furthercomprises an additional conjugating moiety. In some instances, theadditional conjugating moiety is an endosomolytic moiety. In some cases,the endosomolytic moiety is a cellular compartmental release component,such as a compound capable of releasing from any of the cellularcompartments known in the art, such as the endosome, lysosome,endoplasmic reticulum (ER), golgi apparatus, microtubule, peroxisome, orother vesicular bodies with the cell. In some cases, the endosomolyticmoiety comprises an endosomolytic polypeptide, an endosomolytic polymer,an endosomolytic lipid, or an endosomolytic small molecule. In somecases, the endosomolytic moiety comprises an endosomolytic polypeptide.In other cases, the endosomolytic moiety comprises an endosomolyticpolymer.

Endosomolytic Polypeptides

In some embodiments, a molecule of Formula (I): A-X—B—Y—C, is furtherconjugated with an endosomolytic polypeptide. In some cases, theendosomolytic polypeptide is a pH-dependent membrane active peptide. Insome cases, the endosomolytic polypeptide is an amphipathic polypeptide.In additional cases, the endosomolytic polypeptide is a peptidomimetic.In some instances, the endosomolytic polypeptide comprises INF,melittin, meucin, or their respective derivatives thereof. In someinstances, the endosomolytic polypeptide comprises INF or itsderivatives thereof. In other cases, the endosomolytic polypeptidecomprises melittin or its derivatives thereof. In additional cases, theendosomolytic polypeptide comprises meucin or its derivatives thereof.

In some instances, INF7 is a 24 residue polypeptide those sequencecomprises CGIFGEIEELIEEGLENLIDWGNA (SEQ ID NO: 1), orGLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO: 2). In some instances, INF7 or itsderivatives comprise a sequence of: GLFEAIEGFIENGWEGMIWDYGSGSCG (SEQ IDNO: 3), GLFEAIEGFIENGWEGMIDGWYG-(PEG)6-NH2 (SEQ ID NO: 4), orGLFEAIEGFIENGWEGMIWDYG-SGSC-K(GalNAc)2 (SEQ ID NO: 5).

In some cases, melittin is a 26 residue polypeptide those sequencecomprises CLIGAILKVLATGLPTLISWIKNKRKQ (SEQ ID NO: 6), orGIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 7). In some instances, melittincomprises a polypeptide sequence as described in U.S. Pat. No.8,501,930.

In some instances, meucin is an antimicrobial peptide (AMP) derived fromthe venom gland of the scorpion Mesobuthus eupeus. In some instances,meucin comprises of meucin-13 those sequence comprises IFGAIAGLLKNIF-NH₂(SEQ ID NO: 8) and meucin-18 those sequence comprises FFGHLFKLATKIIPSLFQ(SEQ ID NO: 9).

In some instances, the endosomolytic polypeptide comprises a polypeptidein which its sequence is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%sequence identity to INF7 or its derivatives thereof, melittin or itsderivatives thereof, or meucin or its derivatives thereof. In someinstances, the endosomolytic moiety comprises INF7 or its derivativesthereof, melittin or its derivatives thereof, or meucin or itsderivatives thereof.

In some instances, the endosomolytic moiety is INF7 or its derivativesthereof. In some cases, the endosomolytic moiety comprises a polypeptidehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1-5. In somecases, the endosomolytic moiety comprises a polypeptide having at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to SEQ ID NO: 1. In some cases, the endosomolyticmoiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 2-5. In some cases, the endosomolytic moiety comprises SEQID NO: 1. In some cases, the endosomolytic moiety comprises SEQ ID NO:2-5. In some cases, the endosomolytic moiety consists of SEQ ID NO: 1.In some cases, the endosomolytic moiety consists of SEQ ID NO: 2-5.

In some instances, the endosomolytic moiety is melittin or itsderivatives thereof. In some cases, the endosomolytic moiety comprises apolypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 6 or7. In some cases, the endosomolytic moiety comprises a polypeptidehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6. In some cases,the endosomolytic moiety comprises a polypeptide having at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NO: 7. In some cases, the endosomolyticmoiety comprises SEQ ID NO: 6. In some cases, the endosomolytic moietycomprises SEQ ID NO: 7. In some cases, the endosomolytic moiety consistsof SEQ ID NO: 6. In some cases, the endosomolytic moiety consists of SEQID NO: 7.

In some instances, the endosomolytic moiety is meucin or its derivativesthereof. In some cases, the endosomolytic moiety comprises a polypeptidehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 8 or 9. In somecases, the endosomolytic moiety comprises a polypeptide having at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to SEQ ID NO: 8. In some cases, the endosomolyticmoiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 9. In some cases, the endosomolytic moiety comprises SEQID NO: 8. In some cases, the endosomolytic moiety comprises SEQ ID NO:9. In some cases, the endosomolytic moiety consists of SEQ ID NO: 8. Insome cases, the endosomolytic moiety consists of SEQ ID NO: 9.

In some instances, the endosomolytic moiety comprises a sequence asillustrated in Table 1.

TABLE 1 SEQ Name Origin Amino Acid Sequence ID NO: Type Pep-1 NLS fromSimian Virus KETWWETWWTEWSQPKKKRKV 10 Primary 40 large antigen andamphipathic Reverse transcriptase of HIV pVEC VE-cadherinLLIILRRRRIRKQAHAHSK 11 Primary amphipathic VT5 Synthetic peptideDPKGDPKGVTVTVTVTVTGKGDP 12 β-sheet KPD amphipathic C105Y 1-antitrypsinCSIPPEVKFNKPFVYLI 13 — Transportan Galanin and mastoparanGWTLNSAGYLLGKINLKALAALA 14 Primary KKIL amphipathic TP10 Galanin andmastoparan AGYLLGKINLKALAALAKKIL 15 Primary amphipathic MPG A hydrofobicdomain GALFLGFLGAAGSTMGA 16 β-sheet from the fusion amphipathic sequenceof HIV gp4l and NLS of SV40 T antigen gH625 Glycoprotein gH ofHGLASTLTRWAHYNALIRAF 17 Secondary HSV type I amphipathic α-helical CADYPPTG1 peptide GLWRALWRLLRSLWRLLWRA 18 Secondary amphipathic α-helicalGALA Synthetic peptide WEAALAEALAEALAEHLAEALAE 19 Secondary ALEALAAamphipathic α-helical INF Influenza HA2 fusion GLFEAIEGFIENGWEGMIDGWYGC20 Secondary peptide amphipathic α-helical/ pH- dependent membraneactive peptide HA2E5- Influenza HA2 subunit GLFGAIAGFIENGWEGMIDGWYG 21Secondary TAT of influenza virus X31 amphipathic strain fusion peptideα-helical/ PH- dependent membrane active peptide HA2- Influenza HA2subunit GLFGAIAGFIENGWEGMIDGRQIKI 22 pH- penetratin of influenza virusX31 WFQNRRMKWKK-amide dependent strain fusion peptide membrane activepeptide HA-K4 Influenza HA2 subunit GLFGAIAGFIENGWEGMIDG- 23 pH- ofinfluenza virus X31 SSKKKK dependent strain fusion peptide membraneactive peptide HA2E4 Influenza HA2 subunit GLFEAIAGFIENGWEGMIDGGGYC 24pH- of influenza virus X31 dependent strain fusion peptide membraneactive peptide H5WYG HA2 analogue GLFHAIAHFIHGGWHGLIHGWYG 25 pH-dependent membrane active peptide GALA- INF3 fusion peptideGLFEAIEGFIENGWEGLAEALAEAL 26 pH- INF3- EALAA-(PEG)6-NH2 dependent(PEG)6-NH membrane active peptide CM18- Cecropin-A-Melittin₂₋₁₂KWKLFKKIGAVLKVLTTG- 27 pH- TAT11 (CM₁₈) fusion peptide YGRKKRRQRRRdependent membrane active peptide

In some cases, the endosomolytic moiety comprises a Bak BH3 polypeptidewhich induces apoptosis through antagonization of suppressor targetssuch as Bcl-2 and/or Bcl-xL. In some instances, the endosomolytic moietycomprises a Bak BH3 polypeptide described in Albarran, et al.,“Efficient intracellular delivery of a pro-apoptotic peptide with apH-responsive carrier,” Reactive & Functional Polymers 71: 261-265(2011).

In some instances, the endosomolytic moiety comprises a polypeptide(e.g., a cell-penetrating polypeptide) as described in PCT PublicationNos. WO2013/166155 or WO2015/069587.

Linkers

In some embodiments, a linker described herein is a cleavable linker ora non-cleavable linker. In some instances, the linker is a cleavablelinker. In other instances, the linker is a non-cleavable linker.

In some cases, the linker is a non-polymeric linker. A non-polymericlinker refers to a linker that does not contain a repeating unit ofmonomers generated by a polymerization process. Exemplary non-polymericlinkers include, but are not limited to, C₁-C₆ alkyl group (e.g., a C₅,C₄, C₃, C₂, or C₁ alkyl group), homobifunctional cross linkers,heterobifunctional cross linkers, peptide linkers, traceless linkers,self-immolative linkers, maleimide-based linkers, or combinationsthereof. In some cases, the non-polymeric linker comprises a C₁-C₆ alkylgroup (e.g., a C₅, C₄, C₃, C₂, or C₁ alkyl group), a homobifunctionalcross linker, a heterobifunctional cross linker, a peptide linker, atraceless linker, a self-immolative linker, a maleimide-based linker, ora combination thereof. In additional cases, the non-polymeric linkerdoes not comprise more than two of the same type of linkers, e.g., morethan two homobifunctional cross linkers, or more than two peptidelinkers. In further cases, the non-polymeric linker optionally comprisesone or more reactive functional groups.

In some instances, the non-polymeric linker does not encompass a polymerthat is described above. In some instances, the non-polymeric linkerdoes not encompass a polymer encompassed by the polymer moiety C. Insome cases, the non-polymeric linker does not encompass a polyalkyleneoxide (e.g., PEG). In some cases, the non-polymeric linker does notencompass a PEG.

In some instances, the linker comprises a homobifunctional linker.Exemplary homobifunctional linkers include, but are not limited to,Lomant's reagent dithiobis (succinimidylpropionate) DSP,3′3′-dithiobis(sulfosuccinimidyl proprionate (DTSSP), disuccinimidylsuberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyltartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethyleneglycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG),N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA),dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS),dimethyl-3,3′-dithiobispropionimidate (DTBP),1,4-di-3′-(2′-pyridyldithio)propionamido)butane (DPDPB),bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), suchas e.g. 1,5-difluoro-2,4-dinitrobenzene or1,3-difluoro-4,6-dinitrobenzene, 4,4′-difluoro-3,3′-dinitrophenylsulfone(DFDNPS), bis-[(β-(4-azidosalicylamido)ethyl]disulfide (BASED),formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipicacid dihydrazide, carbohydrazide, o-toluidine, 3,3′-dimethylbenzidine,benzidine, α,α′-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid,N,N′-ethylene-bis(iodoacetamide), orN,N′-hexamethylene-bis(iodoacetamide).

In some embodiments, the linker comprises a heterobifunctional linker.Exemplary heterobifunctional linker include, but are not limited to,amine-reactive and sulfhydryl cross-linkers such as N-succinimidyl3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble-long-chainN-succinimidyl 3-(2-pyridyldithio) propionate (sulfo-LC-sPDP),succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT),sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate(sulfo-LC-sMPT),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC),sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs),m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs),N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB),sulfosuccinimidyl(4-iodoacteyl)aminobenzoate (sulfo-sIAB),succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB),sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB),N-(γ-maleimidobutyryloxy)succinimide ester (GMBs),N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs),succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC),succinimidyl6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino) hexanoate(sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive andsulfhydryl-reactive cross-linkers such as 4-(4-N-maleimidophenyl)butyricacid hydrazide (MPBH),4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H),3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactive andphotoreactive cross-linkers such asN-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA),N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA),sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA),sulfosuccinimidyl-2-(ρ-azidosalicylamido)ethyl-1,3′-dithiopropionate(sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB),N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB),N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate (sANPAH),sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate(sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs),sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-dithiopropionate(sAND), N-succinimidyl-4(4-azidophenyl)1,3′-dithiopropionate (sADP),N-sulfosuccinimidyl(4-azidophenyl)-1,3′-dithiopropionate (sulfo-sADP),sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB),sulfosuccinimidyl2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate(sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate(sulfo-sAMCA), ρ-nitrophenyl diazopyruvate (ρNPDP),ρ-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP),sulfhydryl-reactive and photoreactive cross-linkers such as1-(ρ-Azidosalicylamido)-4-(iodoacetamido)butane (AsIB),N-[4-(ρ-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide(APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimidecarbonyl-reactive and photoreactive cross-linkers such as ρ-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive cross-linkerssuch as 4-(ρ-azidosalicylamido)butylamine (AsBA), and arginine-reactiveand photoreactive cross-linkers such as ρ-azidophenyl glyoxal (APG).

In some instances, the linker comprises a reactive functional group. Insome cases, the reactive functional group comprises a nucleophilic groupthat is reactive to an electrophilic group present on a binding moiety.Exemplary electrophilic groups include carbonyl groups-such as aldehyde,ketone, carboxylic acid, ester, amide, enone, acyl halide or acidanhydride. In some embodiments, the reactive functional group isaldehyde. Exemplary nucleophilic groups include hydrazide, oxime, amino,hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

In some embodiments, the linker comprises a maleimide group. In someinstances, the maleimide group is also referred to as a maleimidespacer. In some instances, the maleimide group further encompasses acaproic acid, forming maleimidocaproyl (mc). In some cases, the linkercomprises maleimidocaproyl (mc). In some cases, the linker ismaleimidocaproyl (mc). In other instances, the maleimide group comprisesa maleimidomethyl group, such assuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) orsulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-sMCC) described above.

In some embodiments, the maleimide group is a self-stabilizingmaleimide. In some instances, the self-stabilizing maleimide utilizesdiaminopropionic acid (DPR) to incorporate a basic amino group adjacentto the maleimide to provide intramolecular catalysis of tiosuccinimidering hydrolysis, thereby eliminating maleimide from undergoing anelimination reaction through a retro-Michael reaction. In someinstances, the self-stabilizing maleimide is a maleimide group describedin Lyon, et al., “Self-hydrolyzing maleimides improve the stability andpharmacological properties of antibody-drug conjugates,” Nat.Biotechnol. 32(10):1059-1062 (2014). In some instances, the linkercomprises a self-stabilizing maleimide. In some instances, the linker isa self-stabilizing maleimide.

In some embodiments, the linker comprises a peptide moiety. In someinstances, the peptide moiety comprises at least 2, 3, 4, 5, 6, 7, 8, ormore amino acid residues. In some instances, the peptide moiety is acleavable peptide moiety (e.g., either enzymatically or chemically). Insome instances, the peptide moiety is a non-cleavable peptide moiety. Insome instances, the peptide moiety comprises Val-Cit(valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO: 973), Phe-Lys, Val-Lys,Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit,Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 974), orGly-Phe-Leu-Gly (SEQ ID NO: 975). In some instances, the linkercomprises a peptide moiety such as: Val-Cit (valine-citrulline),Gly-Gly-Phe-Gly (SEQ ID NO: 973), Phe-Lys, Val-Lys, Gly-Phe-Lys,Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit,Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 974), or Gly-Phe-Leu-Gly(SEQ ID NO: 975). In some cases, the linker comprises Val-Cit. In somecases, the linker is Val-Cit.

In some embodiments, the linker comprises a benzoic acid group, or itsderivatives thereof. In some instances, the benzoic acid group or itsderivatives thereof comprise paraaminobenzoic acid (PABA). In someinstances, the benzoic acid group or its derivatives thereof comprisegamma-aminobutyric acid (GABA).

In some embodiments, the linker comprises one or more of a maleimidegroup, a peptide moiety, and/or a benzoic acid group, in anycombination. In some embodiments, the linker comprises a combination ofa maleimide group, a peptide moiety, and/or a benzoic acid group. Insome instances, the maleimide group is maleimidocaproyl (mc). In someinstances, the peptide group is val-cit. In some instances, the benzoicacid group is PABA. In some instances, the linker comprises a mc-val-citgroup. In some cases, the linker comprises a val-cit-PABA group. Inadditional cases, the linker comprises a mc-val-cit-PABA group.

In some embodiments, the linker is a self-immolative linker or aself-elimination linker. In some cases, the linker is a self-immolativelinker. In other cases, the linker is a self-elimination linker (e.g., acyclization self-elimination linker). In some instances, the linkercomprises a linker described in U.S. Pat. No. 9,089,614 or PCTPublication No. WO2015038426.

In some embodiments, the linker is a dendritic type linker. In someinstances, the dendritic type linker comprises a branching,multifunctional linker moiety. In some instances, the dendritic typelinker is used to increase the molar ratio of polynucleotide B to thebinding moiety A. In some instances, the dendritic type linker comprisesPAMAM dendrimers.

In some embodiments, the linker is a traceless linker or a linker inwhich after cleavage does not leave behind a linker moiety (e.g., anatom or a linker group) to a binding moiety A, a polynucleotide B, apolymer C, or an endosomolytic moiety D. Exemplary traceless linkersinclude, but are not limited to, germanium linkers, silicium linkers,sulfur linkers, selenium linkers, nitrogen linkers, phosphorus linkers,boron linkers, chromium linkers, or phenylhydrazide linker. In somecases, the linker is a traceless aryl-triazene linker as described inHejesen, et al., “A traceless aryl-triazene linker for DNA-directedchemistry,” Org Biomol Chem 11(15): 2493-2497 (2013). In some instances,the linker is a traceless linker described in Blaney, et al., “Tracelesssolid-phase organic synthesis,” Chem. Rev. 102: 2607-2024 (2002). Insome instances, a linker is a traceless linker as described in U.S. Pat.No. 6,821,783.

In some instances, the linker is a linker described in U.S. Pat. Nos.6,884,869; 7,498,298; 8,288,352; 8,609,105; or 8,697,688; U.S. PatentPublication Nos. 2014/0127239; 2013/028919; 2014/286970; 2013/0309256;2015/037360; or 2014/0294851; or PCT Publication Nos. WO2015057699;WO2014080251; WO2014197854; WO2014145090; or WO2014177042.

In some embodiments, X, Y, and L are independently a bond or a linker.In some instances, X, Y, and L are independently a bond. In some cases,X, Y, and L are independently a linker.

In some instances, X is a bond or a linker, e.g., a non-polymericlinker. In some instances, X is a bond. In some instances, X isanon-polymeric linker. In some instances, the non-polymeric linker is aC₁-C₆ alkyl group. In some cases, X is a C₁-C₆ alkyl group, such as forexample, a C₅, C₄, C₃, C₂, or C₁ alkyl group. In some cases, the C₁-C₆alkyl group is an unsubstituted C₁-C₆ alkyl group. As used in thecontext of anon-polymeric linker, and in particular in the context of X,alkyl means a saturated straight or branched hydrocarbon radicalcontaining up to six carbon atoms. In some instances, X includes ahomobifunctional linker or a heterobifunctional linker described supra.In some cases, X includes a heterobifunctional linker. In some cases, Xincludes sMCC. In other instances, X includes a heterobifunctionallinker optionally conjugated to a C₁-C₆ alkyl group. In other instances,X includes sMCC optionally conjugated to a C₁-C₆ alkyl group. Inadditional instances, X does not encompass a polymer encompassed by thepolymer moiety C, e.g., X does not encompass a polyalkylene oxide (e.g.,a PEG molecule).

In some instances, Y is a bond or a linker, e.g., a non-polymericlinker. In some instances, Y is a bond. In other cases, Y is anon-polymeric linker. In some embodiments, Y is a C₁-C₆ alkyl group. Insome instances, Y is a homobifunctional linker or a heterobifunctionallinker described supra. In some instances, Y is a homobifunctionallinker described supra. In some instances, Y is a heterobifunctionallinker described supra. In some instances, Y comprises a maleimidegroup, such as maleimidocaproyl (mc) or a self-stabilizing maleimidegroup described above. In some instances, Y comprises a peptide moiety,such as Val-Cit. In some instances, Y comprises a benzoic acid group,such as PABA. In additional instances, Y comprises a combination of amaleimide group, a peptide moiety, and/or a benzoic acid group. Inadditional instances, Y comprises a me group. In additional instances, Ycomprises a me-val-cit group. In additional instances, Y comprises aval-cit-PABA group. In additional instances, Y comprises amc-val-cit-PABA group. In some cases, Y does not encompass a polymerencompassed by the polymer moiety C, e.g., Y does not encompass apolyalkylene oxide (e.g., a PEG molecule).

In some instances, L is a bond or a linker, optionally a non-polymericlinker. In some cases, L is a bond. In other cases, L is a linker,optionally anon-polymeric linker. In some embodiments, L is a C₁-C₆alkyl group. In some instances, L is a homobifunctional linker or aheterobifunctional linker described supra. In some instances, L is ahomobifunctional linker described supra. In some instances, L is aheterobifunctional linker described supra. In some instances, Lcomprises a maleimide group, such as maleimidocaproyl (mc) or aself-stabilizing maleimide group described above. In some instances, Lcomprises a peptide moiety, such as Val-Cit. In some instances, Lcomprises a benzoic acid group, such as PABA. In additional instances, Lcomprises a combination of a maleimide group, a peptide moiety, and/or abenzoic acid group. In additional instances, L comprises a me group. Inadditional instances, L comprises a mc-val-cit group. In additionalinstances, L comprises a val-cit-PABA group. In additional instances, Lcomprises a mc-val-cit-PABA group. In some cases, L, when optionally asa non-polymeric linker, does not encompass a polymer encompassed by thepolymer moiety C, e.g., Y does not encompass a polyalkylene oxide (e.g.,a PEG molecule).

Pharmaceutical Formulation

In some embodiments, the pharmaceutical formulations described hereinare administered to a subject by multiple administration routes,including but not limited to, parenteral (e.g., intravenous,subcutaneous, intramuscular), oral, intranasal, buccal, rectal, ortransdermal administration routes. In some instances, the pharmaceuticalcomposition describe herein is formulated for parenteral (e.g.,intravenous, subcutaneous, intramuscular, intra-arterial,intraperitoneal, intrathecal, intracerebral, intracerebroventricular, orintracranial) administration. In other instances, the pharmaceuticalcomposition describe herein is formulated for oral administration. Instill other instances, the pharmaceutical composition describe herein isformulated for intranasal administration.

In some embodiments, the pharmaceutical formulations include, but arenot limited to, aqueous liquid dispersions, self-emulsifyingdispersions, solid solutions, liposomal dispersions, aerosols, soliddosage forms, powders, immediate release formulations, controlledrelease formulations, fast melt formulations, tablets, capsules, pills,delayed release formulations, extended release formulations, pulsatilerelease formulations, multiparticulate formulations (e.g., nanoparticleformulations), and mixed immediate and controlled release formulations.

In some instances, the pharmaceutical formulation includesmultiparticulate formulations. In some instances, the pharmaceuticalformulation includes nanoparticle formulations. In some instances,nanoparticles comprise cMAP, cyclodextrin, or lipids. In some cases,nanoparticles comprise solid lipid nanoparticles, polymericnanoparticles, self-emulsifying nanoparticles, liposomes,microemulsions, or micellar solutions. Additional exemplarynanoparticles include, but are not limited to, paramagneticnanoparticles, superparamagnetic nanoparticles, metal nanoparticles,fullerene-like materials, inorganic nanotubes, dendrimers (such as withcovalently attached metal chelates), nanofibers, nanohorns, nano-onions,nanorods, nanoropes and quantum dots. In some instances, a nanoparticleis a metal nanoparticle, e.g., a nanoparticle of scandium, titanium,vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc,yttrium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium,silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium,platinum, gold, gadolinium, aluminum, gallium, indium, tin, thallium,lead, bismuth, magnesium, calcium, strontium, barium, lithium, sodium,potassium, boron, silicon, phosphorus, germanium, arsenic, antimony, andcombinations, alloys or oxides thereof.

In some instances, a nanoparticle includes a core or a core and a shell,as in a core-shell nanoparticle.

In some instances, a nanoparticle is further coated with molecules forattachment of functional elements (e.g., with one or more of apolynucleic acid molecule or binding moiety described herein). In someinstances, a coating comprises chondroitin sulfate, dextran sulfate,carboxymethyl dextran, alginic acid, pectin, carragheenan, fucoidan,agaropectin, porphyran, karaya gum, gellan gum, xanthan gum, hyaluronicacids, glucosamine, galactosamine, chitin (or chitosan), polyglutamicacid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease,trypsinogen, chymotrypsinogen, α-chymotrypsin, polylysine, polyarginine,histone, protamine, ovalbumin or dextrin or cyclodextrin. In someinstances, a nanoparticle comprises a graphene-coated nanoparticle.

In some cases, a nanoparticle has at least one dimension of less thanabout 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm.

In some instances, the nanoparticle formulation comprises paramagneticnanoparticles, superparamagnetic nanoparticles, metal nanoparticles,fullerene-like materials, inorganic nanotubes, dendrimers (such as withcovalently attached metal chelates), nanofibers, nanohorns, nano-onions,nanorods, nanoropes or quantum dots. In some instances, a polynucleicacid molecule or a binding moiety described herein is conjugated eitherdirectly or indirectly to the nanoparticle. In some instances, at least1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more polynucleicacid molecules or binding moieties described herein are conjugatedeither directly or indirectly to a nanoparticle.

In some embodiments, the pharmaceutical formulation comprise a deliveryvector, e.g., a recombinant vector, the delivery of the polynucleic acidmolecule into cells. In some instances, the recombinant vector is DNAplasmid. In other instances, the recombinant vector is a viral vector.Exemplary viral vectors include vectors derived from adeno-associatedvirus, retrovirus, adenovirus, or alphavirus. In some instances, therecombinant vectors capable of expressing the polynucleic acid moleculesprovide stable expression in target cells. In additional instances,viral vectors are used that provide for transient expression ofpolynucleic acid molecules.

In some embodiments, the pharmaceutical formulations include a carrieror carrier materials selected on the basis of compatibility with thecomposition disclosed herein, and the release profile properties of thedesired dosage form. Exemplary carrier materials include, e.g., binders,suspending agents, disintegration agents, filling agents, surfactants,solubilizers, stabilizers, lubricants, wetting agents, diluents, and thelike. Pharmaceutically compatible carrier materials include, but are notlimited to, acacia, gelatin, colloidal silicon dioxide, calciumglycerophosphate, calcium lactate, maltodextrin, glycerine, magnesiumsilicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters,sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine,sodium chloride, tricalcium phosphate, dipotassium phosphate, celluloseand cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan,monoglyceride, diglyceride, pregelatinized starch, and the like. See,e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed(Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins 1999).

In some instances, the pharmaceutical formulations further include pHadjusting agents or buffering agents which include acids such as acetic,boric, citric, lactic, phosphoric and hydrochloric acids; bases such assodium hydroxide, sodium phosphate, sodium borate, sodium citrate,sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; andbuffers such as citrate/dextrose, sodium bicarbonate and ammoniumchloride. Such acids, bases and buffers are included in an amountrequired to maintain pH of the composition in an acceptable range.

In some instances, the pharmaceutical formulation includes one or moresalts in an amount required to bring osmolality of the composition intoan acceptable range. Such salts include those having sodium, potassiumor ammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

In some instances, the pharmaceutical formulations further includediluent which are used to stabilize compounds because they provide amore stable environment. Salts dissolved in buffered solutions (whichalso provide pH control or maintenance) are utilized as diluents in theart, including, but not limited to a phosphate buffered saline solution.In certain instances, diluents increase bulk of the composition tofacilitate compression or create sufficient bulk for homogenous blendfor capsule filling. Such compounds include e.g., lactose, starch,mannitol, sorbitol, dextrose, microcrystalline cellulose such asAvicel®; dibasic calcium phosphate, dicalcium phosphate dihydrate;tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-driedlactose; pregelatinized starch, compressible sugar, such as Di-Pac®(Amstar); mannitol, hydroxypropylmethylcellulose,hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents,confectioner's sugar; monobasic calcium sulfate monohydrate, calciumsulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzedcereal solids, amylose; powdered cellulose, calcium carbonate; glycine,kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

In some cases, the pharmaceutical formulations include disintegrationagents or disintegrants to facilitate the breakup or disintegration of asubstance. The term “disintegrate” include both the dissolution anddispersion of the dosage form when contacted with gastrointestinalfluid. Examples of disintegration agents include a starch, e.g., anatural starch such as corn starch or potato starch, a pregelatinizedstarch such as National 1551 or Amijel®, or sodium starch glycolate suchas Promogel® or Explotab®, a cellulose such as a wood product,methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel®PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, andSolka-Floc®, methylcellulose, croscarmellose, or a cross-linkedcellulose, such as cross-linked sodium carboxymethylcellulose(Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linkedcroscarmellose, a cross-linked starch such as sodium starch glycolate, across-linked polymer such as crospovidone, a cross-linkedpolyvinylpyrrolidone, alginate such as alginic acid or a salt of alginicacid such as sodium alginate, a clay such as Veegum® HV (magnesiumaluminum silicate), a gum such as agar, guar, locust bean, Karaya,pectin, or tragacanth, sodium starch glycolate, bentonite, a naturalsponge, a surfactant, a resin such as a cation-exchange resin, citruspulp, sodium lauryl sulfate, sodium lauryl sulfate in combinationstarch, and the like.

In some instances, the pharmaceutical formulations include fillingagents such as lactose, calcium carbonate, calcium phosphate, dibasiccalcium phosphate, calcium sulfate, microcrystalline cellulose,cellulose powder, dextrose, dextrates, dextran, starches, pregelatinizedstarch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride,polyethylene glycol, and the like.

Lubricants and glidants are also optionally included in thepharmaceutical formulations described herein for preventing, reducing orinhibiting adhesion or friction of materials. Exemplary lubricantsinclude, e.g., stearic acid, calcium hydroxide, talc, sodium stearylfumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetableoil such as hydrogenated soybean oil (Sterotex®), higher fatty acids andtheir alkali-metal and alkaline earth metal salts, such as aluminum,calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol,talc, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate,sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or amethoxypolyethylene glycol such as Carbowax™, sodium oleate, sodiumbenzoate, glyceryl behenate, polyethylene glycol, magnesium or sodiumlauryl sulfate, colloidal silica such as Syloid™, Cab-O-Sil®, a starchsuch as corn starch, silicone oil, a surfactant, and the like.

Plasticizers include compounds used to soften the microencapsulationmaterial or film coatings to make them less brittle. Suitableplasticizers include, e.g., polyethylene glycols such as PEG 300, PEG400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propyleneglycol, oleic acid, triethyl cellulose and triacetin. Plasticizers alsofunction as dispersing agents or wetting agents.

Solubilizers include compounds such as triacetin, triethylcitrate, ethyloleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate,vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone,N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropylalcohol, cholesterol, bile salts, polyethylene glycol 200-600,glycofurol, transcutol, propylene glycol, and dimethyl isosorbide andthe like.

Stabilizers include compounds such as any antioxidation agents, buffers,acids, preservatives and the like.

Suspending agents include compounds such as polyvinylpyrrolidone, e.g.,polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetatecopolymer (S630), polyethylene glycol, e.g., the polyethylene glycol hasa molecular weight of about 300 to about 6000, or about 3350 to about4000, or about 7000 to about 5400, sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcelluloseacetate stearate, polysorbate-80, hydroxyethylcellulose, sodiumalginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum,xanthans, including xanthan gum, sugars, cellulosics, such as, e.g.,sodium carboxymethylcellulose, methylcellulose, sodiumcarboxymethylcellulose, hydroxypropylmethylcellulose,hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylatedsorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone andthe like.

Surfactants include compounds such as sodium lauryl sulfate, sodiumdocusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitanmonooleate, polyoxyethylene sorbitan monooleate, polysorbates,polaxomers, bile salts, glyceryl monostearate, copolymers of ethyleneoxide and propylene oxide, e.g., Pluronic® (BASF), and the like.Additional surfactants include polyoxyethylene fatty acid glycerides andvegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; andpolyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,octoxynol 40. Sometimes, surfactants is included to enhance physicalstability or for other purposes.

Viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum,carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, hydroxypropylmethyl cellulose acetate stearate,hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol,alginates, acacia, chitosans and combinations thereof.

Wetting agents include compounds such as oleic acid, glycerylmonostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamineoleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate,sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium saltsand the like.

Therapeutic Regimens

In some embodiments, the pharmaceutical compositions described hereinare administered for therapeutic applications. In some embodiments, thepharmaceutical composition is administered once per day, twice per day,three times per day or more. The pharmaceutical composition isadministered daily, every day, every alternate day, five days a week,once a week, every other week, two weeks per month, three weeks permonth, once a month, twice a month, three times per month, or more. Thepharmaceutical composition is administered for at least 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, ormore.

In some embodiments, one or more pharmaceutical compositions areadministered simultaneously, sequentially, or at an interval period oftime. In some embodiments, one or more pharmaceutical compositions areadministered simultaneously. In some cases, one or more pharmaceuticalcompositions are administered sequentially. In additional cases, one ormore pharmaceutical compositions are administered at an interval periodof time (e.g., the first administration of a first pharmaceuticalcomposition is on day one followed by an interval of at least 1, 2, 3,4, 5, or more days prior to the administration of at least a secondpharmaceutical composition).

In some embodiments, two or more different pharmaceutical compositionsare coadministered. In some instances, the two or more differentpharmaceutical compositions are coadministered simultaneously. In somecases, the two or more different pharmaceutical compositions arecoadministered sequentially without a gap of time betweenadministrations. In other cases, the two or more differentpharmaceutical compositions are coadministered sequentially with a gapof about 0.5 hour, 1 hour, 2 hour, 3 hour, 12 hours, 1 day, 2 days, ormore between administrations.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the composition is given continuously;alternatively, the dose of the composition being administered istemporarily reduced or temporarily suspended for a certain length oftime (i.e., a “drug holiday”). In some instances, the length of the drugholiday varies between 2 days and 1 year, including by way of exampleonly, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days,15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320days, 350 days, or 365 days. The dose reduction during a drug holiday isfrom 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, can be reduced, as a function ofthe symptoms, to a level at which the improved disease, disorder orcondition is retained.

In some embodiments, the amount of a given agent that correspond to suchan amount varies depending upon factors such as the particular compound,the severity of the disease, the identity (e.g., weight) of the subjector host in need of treatment, but nevertheless is routinely determinedin a manner known in the art according to the particular circumstancessurrounding the case, including, e.g., the specific agent beingadministered, the route of administration, and the subject or host beingtreated. In some instances, the desired dose is conveniently presentedin a single dose or as divided doses administered simultaneously (orover a short period of time) or at appropriate intervals, for example astwo, three, four or more sub-doses per day.

The foregoing ranges are merely suggestive, as the number of variablesin regard to an individual treatment regime is large, and considerableexcursions from these recommended values are not uncommon. Such dosagesis altered depending on a number of variables, not limited to theactivity of the compound used, the disease or condition to be treated,the mode of administration, the requirements of the individual subject,the severity of the disease or condition being treated, and the judgmentof the practitioner.

In some embodiments, toxicity and therapeutic efficacy of suchtherapeutic regimens are determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, including, but notlimited to, the determination of the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between the toxic and therapeuticeffects is the therapeutic index and it is expressed as the ratiobetween LD50 and ED50. Compounds exhibiting high therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesare used in formulating a range of dosage for use in human. The dosageof such compounds lies preferably within a range of circulatingconcentrations that include the ED50 with minimal toxicity. The dosagevaries within this range depending upon the dosage form employed and theroute of administration utilized.

Kits/Article of Manufacture

Disclosed herein, in certain embodiments, are kits and articles ofmanufacture for use with one or more of the compositions and methodsdescribed herein. Such kits include a carrier, package, or containerthat is compartmentalized to receive one or more containers such asvials, tubes, and the like, each of the container(s) comprising one ofthe separate elements to be used in a method described herein. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. In one embodiment, the containers are formed from a variety ofmaterials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, bags, containers, bottles,and any packaging material suitable for a selected formulation andintended mode of administration and treatment.

For example, the container(s) include target nucleic acid moleculedescribed herein. Such kits optionally include an identifyingdescription or label or instructions relating to its use in the methodsdescribed herein.

A kit typically includes labels listing contents and/or instructions foruse, and package inserts with instructions for use. A set ofinstructions will also typically be included.

In one embodiment, a label is on or associated with the container. Inone embodiment, a label is on a container when letters, numbers or othercharacters forming the label are attached, molded or etched into thecontainer itself; a label is associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. In one embodiment, a label is used toindicate that the contents are to be used for a specific therapeuticapplication. The label also indicates directions for use of thecontents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are presented ina pack or dispenser device which contains one or more unit dosage formscontaining a compound provided herein. The pack, for example, containsmetal or plastic foil, such as a blister pack. In one embodiment, thepack or dispenser device is accompanied by instructions foradministration. In one embodiment, the pack or dispenser is alsoaccompanied with a notice associated with the container in formprescribed by a governmental agency regulating the manufacture, use, orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the drug for human or veterinary administration.Such notice, for example, is the labeling approved by the U.S. Food andDrug Administration for prescription drugs, or the approved productinsert. In one embodiment, compositions containing a compound providedherein formulated in a compatible pharmaceutical carrier are alsoprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

Certain Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the claimed subject matter belongs. It is to be understoodthat the foregoing general description and the following detaileddescription are exemplary and explanatory only and are not restrictiveof any subject matter claimed. In this application, the use of thesingular includes the plural unless specifically stated otherwise. Itmust be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. In this application, theuse of “or” means “and/or” unless stated otherwise. Furthermore, use ofthe term “including” as well as other forms, such as “include”,“includes,” and “included,” is not limiting.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 μL” means “about 5 μL” and also “5 μL.” Generally, the term“about” includes an amount that would be expected to be withinexperimental error.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

As used herein, the terms “individual(s)”, “subject(s)” and “patient(s)”mean any mammal. In some embodiments, the mammal is a human. In someembodiments, the mammal is a non-human. None of the terms require or arelimited to situations characterized by the supervision (e.g. constant orintermittent) of a health care worker (e.g. a doctor, a registerednurse, a nurse practitioner, a physician's assistant, an orderly or ahospice worker).

As used herein the terms “DMD,” “DMD gene,” and equivalents thereofrefer to the DMD gene that encodes for the protein dystrophin. Inaddition, the terms “DMD” and “DMD gene” are used interchangeable, andboth terms refer to the dystrophin gene.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1. Antisense Oligonucleotide Sequences and Synthesis

Phosphorodiamidate morpholino oligomers (PMO), phosphorothioateantisense oligonucleotides (PS ASO), and antisense oligonucleotides(ASOs) were synthesized.

The PMO sequence was

5'GGCCAAACCTCGGCTTACCTGAAAT3'Primary amine (SEQ ID NO: 28) and can be seen in FIG. 1 with endnucleotides expanded. The PMO contains a C3-NH₂ conjugation handle atthe 3′ end of the molecule for conjugation. PMOs were fully assembled onsolid phase using standard solid phase synthesis protocols and purifiedover HPLC.

The PS ASO sequence was

(SEQ ID NO: 29) C6-GGCCAAACCUCGGCUUACCU and can be seen in FIGS. 2A-2B with end nucleotides expanded. Thestructure of the PS ASO comprised a phosphate backbone that was 100%phosphorothioate linkages and all the ribose sugars contained a 2′ 2′OMemodification. The PS ASO also contained a C6-NH₂ conjugation handle atthe 5′ end of the molecule for conjugation. The PS ASOs were fullyassembled on the solid phase using standard solid phase phosphoramiditechemistry and purified over HPLC.

ASOs were fully assembled on the solid phase using standard solid phasephosphoramidite chemistry and purified over HPLC. ASOs contained aC6-NH₂ conjugation handle at the 5′ end of the molecule for conjugation.

Example 2. Detection of DMD Exon Skipping

Methods for Determining DMD Exon 23 Skipping in Differentiated C1C12Cells

Mouse myoblast C2C12 cells were plated at 50,000-100,000/well in 24-wellplates in 0.5 mL 10% FBS RPMI 1640 media and incubated at 37° C. with 5%CO₂ overnight. On the second day, cells were switched to differentiationmedia (2% horse serum RPMI 1640 and 1 μM insulin) and incubated for 3-5days. Following incubation, samples were added and incubated for 24hours. After the sample treatment, 1 mL of fresh media (with nocompounds) was changed every day for 2 more days. At 72 hours after thestart of treatments, cells were harvested. RNAs were isolated usingInviTrap RNA Cell HTS 96 Kit (B-Bridge International #7061300400) andreverse transcribed using High Capacity cDNA Reverse transcription Kit(ThermoFisher #4368813). PCR reactions were performed using DreamTaq™PCR Mastermix (ThermoFisher #K1072). The primary PCR used primers inexon 20 (Ex20F 5′-CAGAATTCTGCCAATTGCTGAG) (SEQ ID NO; 30) and exon 26(Ex26R 5′-TTCTTCAGCTTGTGTCATCC) (SEQ ID NO: 31) to amplify both skippedand unskipped molecules using the protocol in Table 2.

TABLE 2 PCR Protocol Hot Start 95°C. for 2 minutes Denaturation 95°C.for 0.5 minute Annealing of primers 50°C. for 0.5 minute Primerextension 72°C. for 1 minute Final extension 72°C. for 5 minutes Numberof Cycles 10

For the nested PCR, primary PCR reactions were diluted with water 100×,and 5 μl was used for nested PCR reaction (50 μl total reaction volume).Nested PCR used primers in exon 20 (Ex20F2: 5′-ACCCAGTCTACCACCCTATC (SEQID NO: 32) and exon 25 (Ex25R: 5′-CTCTTTATCTTCTGCCCACCTT (SEQ ID NO: 33)to amplify both skipped and unskipped molecules using the protocol inTable 3.

TABLE 3 Nested PCR Protocol Hot Start 95°C. for 2 minutes Denaturation95°C. for 0.5 minute Annealing of primers 50°C. for 0.5 minute Primerextension 72°C. for 1 minute Final extension 72°C. for 5 minutes Numberof Cycles 35

PCR reactions were analyzed using 4% TAE agarose gels. The wild-type(WT) DMD product had an expected size of 788 base pairs and the skippedDMD A23 of 575 base pairs.

Animals

All animal studies were conducted following protocols in accordance withthe Institutional Animal Care and Use Committee (IACUC) at ExploraBioLabs, which adhere to the regulations outlined in the USDA AnimalWelfare Act as well as the “Guide for the Care and Use of LaboratoryAnimals” (National Research Council publication, 8th Ed., revised in2011). All mice were obtained from either Charles River Laboratories orHarlan Laboratories.

In Vivo Mouse Model

WT CD-1 mice (4-6 weeks old) were dosed via intravenous (iv) injectionwith the indicated antisense conjugates (ASCs) and doses. The “naked”PMO or ASO were dosed via intramuscular injection at the indicateddoses. After 4, 7, or 14 days, heart and gastrocnemius muscle tissueswere harvested and snap-frozen in liquid nitrogen. RNAs were isolatedwith Trizol and RNeasy Plus 96 Kit (Qiagen, #74192) and reversedtranscribed using High Capacity cDNA Reverse transcription Kit(ThermoFisher #4368813). Nested PCR reactions were performed asdescribed. PCR reactions were analyzed in 4% (or 1%) TAE agarose gelswhich were quantitated by densitometry.

To confirm exon 23 skipping in treated mice, DNA fragments were isolatedfrom the 4% agarose gels and sequenced.

To quantitatively determine the skipped DMD mRNA copy number, qPCRprimer/probe sets were designed to quantify skipped and WT DMD mRNA(FIG. 3). qPCR quantification standards were designed and produced viaPCR using designed PCR primers as seen in Table 4. For the qPCR standardfor WT and DMD, following PCR a 733 base pair fragment was isolated fromthe agarose gel. For qPCR standard for skipped DMA, the nested primerswere used.

The amplification efficiency of the qPCR primer/probes were determinedto be within 10% of expected efficiency. qPCR reactions were performedin QuantStudio 7 and Tagman™ PCR Universal Mastermix II (ThermoFisher#4440041) according to manufacturer's instructions.

TABLE 4 SEQ Primer/ ID NO Probe Sequence DMD Δ-23, 34 Forward5′GCGCTATCAGGA for Ex23 Primer GACAATGAG skipping 35 Reverse5′GTTTTTATGTGA Primer TTCTGTAATTTCCC 36 Probe 5′CTCTCTGTACCTTATCTTAGTGTT DMD Ex22- 37 Forward 5′TGGAGGAGAGAC 23, for WT PrimerTCGGGAAA DMD only 38 Reverse 5′TTGAAGCCATTT Primer TGTTGCTCTTT 39 Probe5′ACAGGCTCTGCA AAGT DMD Ex20- 40 Forward 5′AACAGATGACAA 21, for AllPrimer CTACTGCCGAAA DMD 41 Reverse 5′TTGGCTCTGATA Primer GGGTGGTAGAC 42Probe 5′CTTGTTGAAAAC CC qPCR standard 43 Forward 5′TGAGGGTGTTAA for WTand all Primer TGCTGAAAGTA DMD 44 Reverse 5′CACCAACTGGGA Primer GGAAAGTT

Example 3: Conjugate Synthesis

Analytical and Purification Methods

Analytical and purification methods were performed according to Tables5-11.

TABLE 5 Size exclusion chromatography (SEC) methods Size ExclusionChromatography Mobile (SEC) Method Column Phase Flow Rate method 1 TOSOHBiosciences, 150 mM 1.0 mL/minute TSKgelG3000SW phosphate for 20 minutesXL, 7.8 ×300 mm, 5 buffer μM method 2 TOSOH Biosciences, PBS pH 7.4 1.0mL/minute TSKgelG3000SW, for 180 minutes 21.5 ×600 mm, 5 μM

TABLE 6 Hydrophobic interaction chromatography (HIC) method 1 GradientColumn Column Solvent Volume % A % B GE, HiScreen Solvent A: 50 mMphosphate buffer, 1.00 95 5 Butyl HP, 0.8M Ammonium Sulfate, pH 7.0 30 0100 4.7 mL Solvent B: 80% 50 mM phosphate 5 0 100 buffer, 20% IPA, pH7.0 Flow Rate: 1.0 mL/minute

TABLE 7 Hydrophobic interaction chromatography (HIC) method 2 GradientColumn Solvent Time % A % B Thermo Scientific, Solvent A: 100 mMphosphate 0.00 100 0 MAbPac HIC-20, buffer, 1.8M Ammonium 2.00 100 0 4.6mm ID × Sulfate, pH 7.0 22.00 0 100 10 cm, 5 um Solvent B: 80% 100 mM25.00 0 100 phosphate buffer, 20% IPA, 26.00 100 0 pH 7.0 30.00 100 0Flow Rate: 0.7 mL/minute

TABLE 8 Hydrophobic interaction chromatography (HIC) method 3 GradientColumn % % Column Solvent Volume A B GE, HiScreen Solvent A: 50 mMphosphate buffer, 1 100 0 Butyl HP, 4.7 mL 0.8M Ammonium Sulfate, pH 7.025 0 80 Solvent B: 80% 50 mM phosphate 1 0 100 buffer, 20% IPA, pH 7.0 20 100 Flow Rate: 1.0 mL/minute

TABLE 9 Hydrophobic interaction chromatography (HIC) method 4 GradientColumn Solvent Time % A % B Thermo Scientific, Solvent A: 100 mMphosphate 0.00 100 0 MAbPac HIC-20, buffer, 1.8M Ammonium 5.00 100 0 4.6mm ID ×10 Sulfate, pH 7.0 20.00 0 100 cm, 5 um Solvent B: 80% 100 mM25.00 0 100 phosphate buffer, 20% IPA, 26.00 100 0 pH 7.0 30.00 100 0Flow Rate: 0.5 mL/minute

TABLE 10 Strong anion exchange chromatography (SAX) method 1 GradientColumn Column Solvent Volume % A % B Tosoh Bioscience, Solvent A: 20 mMTRIS buffer, 0.5 100 0 TSKGel SuperQ- pH 8.0; Solvent B: 20 mM TRIS, 0.580 20 5PW, 21.5 mm 1.5M NaCl, pH 8.0 17 20 80 ID ×15 cm, Flow Rate: 6.0mL/minute 0.5 0 100 13 um 0.5 0 100

TABLE 11 Strong anion exchange chromatography (SAX) method 2 GradientColumn Solvent Time % A % B Thermo Scientific, Solvent A: 80% 10 mM TRIS0.0 90 10 ProPac ™SAX-10, pH 8, 20% ethanol 3.00 90 10 Bio LC ™, 4 ×250Solvent B: 80% 10 mM TRIS 17.00 0 100 mm pH 8, 20% ethanol, 1.5M NaCl21.00 0 100 Flow Rate: 0.75 mL/minute 22.00 90 10 25.00 90 10

Anti-Transferrin Receptor Antibody

Anti-mouse transferrin receptor antibody or anti-CD71 mAb that was usedwas a rat IgG2a subclass monoclonal antibody that binds mouse CD71 ormouse transferrin receptor 1 (mTfR1). The antibody was produced byBioXcell and it is commercially available (Catalog #BE0175).

Anti-CD71 Antibody Morpholino Antisense Oligonucleotide Conjugate(Anti-CD71 mAb-PMO)

Anti-CD71 mAb-PMO Conjugation

Anti-CD71 antibody (10 mg/mL) in borate buffer (25 mM sodiumtetraborate, 25 mM NaCl, 1 mM Diethylene triamine pentaacetic acid, pH8.0) was reduced by adding 4 equivalents oftris(2-carboxyethyl)phosphine (TCEP) in water and incubating at 37° C.for 4 hours. 4(N-Maleimidomethyl)cyclohexanecarboxylic acidN-hydroxysuccinimide ester (SMCC) was coupled to the primary amine onthe 3′ end of the phosphorodiamidate morpholino oligomer (PMO) byincubating the PMO (50 mg/mL) in DMSO with 10 equivalents of SMCC (10mg/mL) in DMSO for one hour. Unconjugated SMCC was removed byultrafiltration using Amicon Ultra-15 centrifugal filter units with aMWCO of 3 kDa. The PMO-SMCC was washed three times with acetate buffer(10 mM sodium acetate, pH 6.0) and used immediately. The reducedantibody was mixed with 2.25 equivalents of PMO-SMCC and incubatedovernight at 4° C. The pH of the reaction mixture was then reduced to7.5, and 8 equivalents of N-Ethylmaleimide was added to the mixture atroom temperature for 30 minutes to quench unreacted cysteines. Analysisof the reaction mixture by hydrophobic interaction chromatography (HIC)method 2 showed antibody-PMO conjugates along with unreacted antibodyand PMO (FIG. 4). FIG. 4 shows a chromatogram of anti-CD71 mAb-PMOreaction mixture produced with HIC method 2 showing free antibody peak(1), free PMO (2), DAR 1 (3), DAR 2 (4), DAR 3 (5), DAR >3 (6). “DAR”refers to a drug-to-antibody ratio. The number in parentheses refers tothe peak in the chromatogram.

Purification

The reaction mixture was purified with an AKTA Explorer FPLC using HICmethod 1. Fractions containing conjugates with a drug to antibody ratioof one (DAR 1) and two (DAR 2) were combined and concentrated withAmicon Ultra-15 centrifugal filter units with a MWCO of 50 kDaseparately from conjugates with a DAR greater than 2. Concentratedconjugates were buffer exchanged with PBS (pH 7.4) using Amicon Ultra-15centrifugal filter units prior to analysis.

Analysis of the Purified Conjugate

The isolated conjugates were characterized by size exclusionchromatography (SEC) and HIC. SEC method 1 was used to confirm theabsence of high molecular weight aggregates and unconjugated PMOs (FIGS.5A-5C). FIG. 5A shows a chromatogram of anti-CD71 mAb produced using SECmethod 1. FIG. 5B shows a chromatogram of anti-CD71 mAb-PMO DAR 1,2produced using SEC method 1. FIG. 5C shows a chromatogram of anti-CD71mAb-PMO DAR greater than 2 produced using SEC method 1. “DAR” refers toa drug-to-antibody ratio.

The purity of the conjugate was assessed by analytical HPLC using HICmethod 2 (FIGS. 6A-6C). FIG. 6A shows a chromatogram of anti-CD71 mAbproduced using HIC method 2. FIG. 6B shows a chromatogram of purifiedanti-CD71 mAb-PMO DAR 1,2 conjugate produced using HIC method 2. FIG. 6Cshows a chromatogram of purified anti-CD71 mAb-PMO DAR >2 conjugateproduced using HIC method 2. The 260/280 nm UV absorbance ratio of eachsample was compared to a standard curve of known ratios of PMO andantibody to confirm DAR. The DAR 1,2 sample had an average DAR of ˜1.6while the DAR greater than 2 sample had an average DAR of ˜3.7. “DAR”refers to a drug-to-antibody ratio.

Anti-CD71 Fab Morpholino Antisense Oligonucleotide Conjugate (Anti-CD71Fab-PMO)

Antibody Digestion with Pepsin

Anti-CD71 antibody (5 mg/mL) in 20 mM acetate buffer (pH 4.0) wasincubated with immobilized pepsin for 3 hours at 37° C. The resin wasremoved and the reaction mixture was washed with PBS (pH 7.4) usingAmicon Ultra-15 centrifugal filter units with a MWCO of 30 kDa. Theretentate was collected and purified using size exclusion chromatography(SEC) method 2 to isolate the F(ab′)2 fragment.

Anti-CD71 (Fab)-PMO Conjugation

The F(ab′)2 fragment (15 mg/mL) in borate buffer (pH 8.0) was reduced byadding 10 equivalents of TCEP in water and incubating at 37° C. for 2hours. SMCC was added to the primary amine on the 3′ end of the PMO byincubating the PMO (50 mg/mL) in DMSO with 10 equivalents of SMCC (10mg/mL) in DMSO for 1 hour. Unconjugated SMCC was removed byultrafiltration using Amicon Ultra-15 centrifugal filter units with aMWCO of 3 kDa. The PMO-SMCC was washed three times with acetate buffer(pH 6.0) and used immediately. The reduced F(ab′) fragment (Fab) wasbuffer exchanged into borate buffer (pH 8.0) using Amicon Ultra-15Centrifugal Filter Units with a MWCO of 10 kDa, and 1.75 equivalents ofPMO-SMCC was added and incubated overnight at 4° C. The pH of thereaction mixture was then reduced to 7.5, and 6 equivalents ofN-Ethylmaleimide was added to the mixture at room temperature for 30minutes to quench unreacted cysteines. Analysis of the reaction mixtureby hydrophobic interaction chromatography (HIC) method 3 showedanti-CD71 (Fab)-PMO conjugates along with unreacted Fab (FIG. 7A). FIG.7A shows a chromatogram of FPLC purification of anti-CD71 Fab-PMO usingHIC method 3.

Purification

The reaction mixture was purified with an AKTA Explorer FPLC using HICmethod 3. Fractions containing conjugates with a DAR of one, two andthree were combined and concentrated separately. Concentrated conjugateswere buffer exchanged with PBS (pH 7.4) using Amicon Ultra-15centrifugal filter units with a MWCO of 10 kDa prior to analysis.

Analysis of the Purified Conjugate

The isolated conjugates were characterized by SEC, and HIC. SEC method 1was used to confirm the absence of high molecular weight aggregates andunconjugated PMO. See FIGS. 7B-7E. FIG. 7B shows a chromatogram ofanti-CD71 Fab produced using SEC method 1. FIG. 7C shows a chromatogramof anti-CD71 Fab-PMO DAR 1 conjugate produced using SEC method 1. FIG.7D shows a chromatogram of anti-CD71 Fab-PMO DAR 2 conjugate producedusing SEC method 1. FIG. 7E shows a chromatogram of anti-CD71 Fab-PMODAR 3 conjugate produced using SEC method 1. The purity of the conjugatewas assessed by analytical HPLC using HIC method 4. See FIGS. 7F-7I.FIG. 7F shows a chromatogram of anti-CD71 Fab produced using HIC method4. FIG. 7G shows a chromatogram of anti-CD71 Fab-PMO DAR 1 conjugateproduced using HIC method 4. FIG. 7H shows a chromatogram of anti-CD71Fab-PMO DAR 2 conjugate produced using HIC method 4. FIG. 7I shows achromatogram of anti-CD71 Fab-PMO DAR 3 conjugate produced using HICmethod 4. “DAR” refers to drug-to-antibody ratio. The 260/280 nm UVabsorbance ratio of each sample was compared to a standard curve ofknown ratios of PMO and Fab to confirm DAR.

Anti-CD71 Antibody Phosphorothioate Antisense Oligonucleotide Conjugate(Anti-CD71 mAb-PS ASO)

Anti-CD71 mAb-PS ASO

Anti-CD71 antibody (10 mg/mL) in borate buffer (pH 8.0) was reduced byadding 4 equivalents of TCEP in water and incubating at 37° C. for 4hours. 4(N-Maleimidomethyl)cyclohexanecarboxylic acidN-hydroxysuccinimide ester (SMCC) was added to the primary amine on the5′ end of the PS-ASO by incubating the PS ASO (50 mg/mL) in 1:1 mixtureof 250 mM PB (pH 7.5) and DMSO with 10 equivalents of SMCC (10 mg/mL) inDMSO for 1 hour. Unconjugated SMCC was removed by ultrafiltration usingAmicon Ultra-15 centrifugal filter units with a MWCO of 3 kDa. The PSASO-SMCC was washed three times with acetate buffer (pH 6.0) and usedimmediately. The reduced antibody was mixed with 1.7 equivalents of PSASO-SMCC and incubated overnight at 4° C. The pH of the reaction mixturewas then reduced to 7.4, and 8 equivalents of N-Ethylmaleimide was addedto the mixture at room temperature for 30 minutes to quench unreactedcysteines. Analysis of the reaction mixture by strong anion exchangechromatography (SAX) method 2 showed antibody-PS ASO conjugates alongwith unreacted antibody and ASO (FIG. 8A). FIG. 8A shows a chromatogramof anti-CD71 mAb-PS ASO reaction mixture produced with SAX method 2showing free antibody peak (1), free PS ASO (5), DAR 1 (2), DAR 2 (3),DAR >2 (4). “DAR” refers to a drug-to-antibody ratio. The number inparentheses refers to the peak.

Purification

The reaction mixture was purified with an AKTA Explorer FPLC using SAXmethod 1. Fractions containing conjugates with a drug-to-antibody ratio(DAR) of one, two and three were combined and concentrated separatelyand buffer exchanged with PBS (pH 7.4) using Amicon Ultra-15 centrifugalfilter units with a MWCO of 50 kDa prior to analysis.

Analysis of the Purified Conjugate

The isolated conjugates were characterized by size exclusionchromatography (SEC) and SAX. Size exclusion chromatography method 1 wasused to confirm the absence of high molecular weight aggregates andunconjugated ASO. See FIGS. 8B-8E. FIG. 8B shows a chromatogram ofanti-CD71 mAb produced using SEC method 1. FIG. 8C shows a chromatogramof anti-CD71 mAb-PS ASO DAR 1 conjugate produced using SEC method 1.FIG. 8D shows a chromatogram of anti-CD71 mAb-PS ASO DAR 2 conjugateproduced using SEC method 1. FIG. 8E shows a chromatogram of anti-CD71mAb-PS ASO DAR 3 conjugate produced using SEC method 1. The purity ofthe conjugate was assessed by analytical HPLC using SAX method 2. SeeFIGS. 8F-8H. FIG. 8F shows a chromatogram of anti-CD71 mAb-PS ASO DAR 1conjugate produced using SAX method 2. FIG. 8G shows a chromatogram ofanti-CD71 mAb-PS ASO DAR 2 conjugate produced using SAX method 2. FIG.8H shows a chromatogram of anti-CD71 mAb-PS ASO DAR 3 conjugate producedusing SAX method 2. The 260/280 nm UV absorbance ratio of each samplewas compared to a standard curve of known ratios of ASO and antibody toconfirm drug-to-antibody ratio (DAR).

Example 4: In Vitro Activity of Anti-CD71 mAb-PMO Conjugate

The anti-CD71 mAb-PMO conjugate was made and characterized as describedin Example 3. The conjugate was assessed for its ability to mediate exonskipping in vitro in differentiated C2C12 cells using nested PCR usingmethods similar to Example 2. Briefly, the potency of “naked” morpholinoASO (“PMO”) was compared to an anti-CD71 mAb-PMO conjugate at multipleconcentrations with the relevant vehicle controls. Controls includedvehicle (“Veh”), scramble morpholino at 50 uM (“Scr50”), and no antibody(“Neg-Ab”). The concentrations of PMO used included 50 uM, 1 uM, and0.02 uM. The concentrations of anti-CD71 mAB-PMO DAR 1,2 used included200 nM, 20 nM, and 2 nM. “DAR” refers to drug-to-antibody ratio.

Following cDNA synthesis, two rounds of PCR amplification (primary andnested PCR) were used to detect exon-skipping. PCR reactions wereanalyzed in a 4% TAE agarose gel (FIG. 9).

Referring to FIG. 9, anti-CD71 mAb-PMO conjugate produced measurableexon 23 skipping in differentiated C2C12 cells and lower concentrationsthan the “naked” PMO control. The wild-type product had an expected sizeof 788 base pairs and the skipped DMD A23 of 575 base pairs.

A second experiment included an anti-CD71 Fab-PMO conjugate and a PMOtargeted with an anti-EGFR (“Z-PMO”) as a negative control (FIG. 10).The concentrations of PMO used included 10 uM and 2 uM. Theconcentrations of anti-CD71 mAb-PMO used included 0.2 uM and 0.04 uM.Anti-CD71 mAb-PMO had a DAR of 2. Z-PMO was used at a concentration of0.2 uM and had a DAR of 2. Concentrations of anti-CD71 Fab-PMO included0.6 uM and 0.12 uM. DAR of 1, 2, and 3 for anti-CD71 mAb-PMO at 0.6 uMand 0.12 uM were assayed.

Referring to FIG. 10, Receptor mediated uptake utilizing the transferrinreceptor, the anti-CD71 mAb-PMO, and anti-CD71 Fab-PMO conjugatesresulted in measurable exon 23 skipping in C2C12 cells and lowerconcentrations than the “naked” PMO control. There was no measurableexon 23 skipping from the Z-PMO at the concentration tested, whichproduced skipping from the anti-CD71 conjugates.

Example 5. In Vitro Activity of Anti-CD71-ASO mAb PS Conjugate

The anti-CD71 mAb-PS ASO conjugate was made and characterized asdescribed in Example 3. The conjugate was assessed for its ability tomediate exon skipping in vitro in differentiated C2C12 cells usingnested PCR using similar methods as described in Example 2. Briefly, thepotency of “naked” phosphorothioate ASO (PS ASO) was compared to ananti-CD71 mAb-PS ASO conjugate at multiple concentrations, with therelevant vehicle control. Two rounds of of PCR amplification (primaryand nested PCR) were performed following cDNA synthesis to detectexon-skipping. PCR reactions were analyzed in a 4% TAE agarose gel (FIG.11). FIG. 11 shows an agarose gel of PMO, ASO, conjugated anti-CD71mAb-ASO of DAR1 (“ASC-DAR1”), conjugated anti-CD71 mAb-ASO of DAR2(“ASC-DAR2”), and conjugated anti-CD71 mAb-ASO of DAR3 (“ASC-DAR3”).“PMO” and “ASO” refers to free PMO and ASO, unconjugated to antibody.“Veh” refers to vehicle only. The concentrations tested included 0.2, 1,and 5 micromolar (μM).

Referring to FIG. 11, the anti-CD71 mAb-PS ASO conjugate producedmeasurable exon 23 skipping in differentiated C2C12 cells and lowerconcentrations than the “naked” PS ASO control. The wild-type producthad an expected size of 788 base pairs and the skipped DMD A23 of 575base pairs.

Example 6: In Vivo Activity of Anti-CD71 mAb-PMO Conjugate

The anti-CD71 mAb-PMO conjugate was made and characterized as describedin Example 3. The conjugate anti-CD71 mAb-PMO DAR1,2 anti-CD71 andmAb-PMO DAR>2 were assessed for its ability to mediate exon skipping invivo in wild-type CD-1 mice using similar methods as described inExample 2. “DAR” refers to drug-to-antibody ratio.

Mice were dosed via intravenous (iv) injection with the mAb, vehiclecontrol, and antisense conjugates (ASCs) at the doses as provided inTable 12. “DAR” refers to drug-to-antibody ratio. The “naked” PMO wasdosed via intramuscular injection into the gastrocnemius muscle at thedoses provided in Table 12. After 4, 7, or 14 days, heart andgastrocnemius muscle tissues were harvested and snap-frozen in liquidnitrogen. RNAs were isolated, reversed transcribed and a nested PCRreactions were performed. PCR reactions were analyzed in 4% TAE agarosegels which were then quantitated by densitometry.

TABLE 12 In vivo study design PMO PMO:mAb Harvest mAb dose Dose RatioTime Group Test Article N (mg/kg) (mg/kg) (mol/mol) (h) 1 anti-CD71 3 504.8 1.6 96 mAb-PMO, DAR1,2 2 anti-CD71 3 50 4.8 1.6 168 mAb-PMO, DAR1,23 anti-CD71 3 50 4.8 1.6 336 mAb-PMO, DAR1,2 4 anti-CD71 3 50 10.5 3.796 mAb-PMO, DAR>2 5 anti-CD71 3 50 10.5 3.7 168 mAb-PMO, DAR>2 6anti-CD71 3 50 10.5 3.7 336 mAb-PMO, DAR>2 7 anti-CD71 mAb 3 50 96 8anti-CD71 mAb 3 50 168 9 anti-CD71 mAb 3 50 336 10 PMO 3 40 ug/inj. 9611 PMO 3 40 ug/inj. 168 12 PMO 3 40 ug/inj. 336 13 Vehicle 3 96 14Vehicle 3 168 15 Vehicle 3 336

FIG. 12A shows a gel electrophoresis of gastrocnemius muscle samplesfrom mice administered anti-CD71 mAb-PMO DAR 1,2, anti-CD71 mAb-PMODAR>2, anti-CD71 mAb, PMO, and vehicle for 4, 7, or 14 days. Thewild-type product had an expected size of 788 base pairs and the skippedDMD A23 of 575 base pairs. Anti-CD71 mAb-PMO DAR 1,2 and anti-CD71mAb-PMO DAR>2 produced measurable exon 23 skipping in gastrocnemiusmuscle and lower concentrations than the “naked” PMO control. Theintensity of the bands on the gel (FIG. 12A) was quantitated bydensitometry as seen in FIG. 12B. FIG. 12C shows the quantification ofin vivo exon skipping in wild-type mice gastrocnemius muscle usingTagman qPCR.

FIG. 13A shows a gel electrophoresis of heart samples from miceadministered anti-CD71 mAb-PMO DAR 1,2, anti-CD71 mAb-PMO DAR>2,anti-CD71 mAb, PMO, and vehicle for 4, 7, or 14 days. The wild-typeproduct had an expected size of 788 base pairs and the skipped DMD A23of 575 base pairs. The intensity of the bands on the gel (FIG. 13A) wasquantitated by densitometry as seen in FIG. 13B. Similar results as withthe gastrocnemius muscle samples were obtained. Anti-CD71 mAb-PMO DAR1,2 and anti-CD71 mAb-PMO DAR>2 produced measurable exon 23 skipping ingastrocnemius muscle and lower concentrations than the “naked” PMOcontrol.

DNA fragments were then isolated from the 4% agarose gels and sequenced.The sequencing data confirmed the correct sequence in the skipped andwild-type products as seen in FIG. 14.

Example 7. Antisense Oligonucleotide Sequences and Synthesis

The sequences in Table 13 were made targeting different exons indifferent genes.

TABLE 13 Sequences SEQ ID NO. Target PMO sequence (5′to 3′) 45 Exon 23in mouse GGCCAAACCTCGGCTTACCTGAA dystrophin AT 46 Exon 2 in mouseAGCCCATCTTCTCCTGGTCCTGG myostatin (MSTN) GAAGG 47 Exon 11 in mouseATCCTCTTTGGTAACCTCACCTC phenylalanine AC hydroxylase (PAH) 48 KRAS-011(human TCGTCCACAAAATGATTCTGAAT cancer) TA 49 ScrambleCGGTGTGTGTATCATTCTCTAGT GT

Example 8. In Vivo Activity of CD71 mAb-PMO Conjugate in MultipleTissues

The CD71 mAb-PMO conjugates were made and characterized as described inExample 3. The conjugate (DAR3+) was assessed for its ability to mediateexon skipping in vivo in wild type CD-1 mice, see example 2 for fullexperimental details. In brief, mice were dosed via intravenous (iv)injection with vehicle control and indicated ASCs at the dosesindicated, see FIG. 7A. After 7, 14 or 28 days, diaphragm, heart andgastrocnemius muscle tissues were harvested and snap-frozen in liquidnitrogen. RNAs were isolated, reversed transcribed, real-time qPCR andnested PCR reactions were performed as described in Example 2 using theappropriate primer/probe sets. PCR reactions were analyzed in 1% TAEagarose gels.

In vivo study design to assess the ability of the CD71 mAb-PMO conjugateto mediate exon 23 skipping in wild type mice is seen in Table 14.

TABLE 14 In vivo study design mAb-PMO PMO PMO:mAb Harvest mAb dose DoseRatio Time Group Test Article N (mg/kg) (mg/kg) (mol/mol) (WEEKS) 1Vehicle 3 1 2 Vehicle 3 4 3 CD71-scr, 3 50 10 3.0 2 DAR3+ 4 CD71-DMD 350 10 3.0 1 PMO, DAR3+ 5 CD71-DMD 3 50 10 3.0 2 PMO, DAR3+ 6 CD71-DMD 350 10 3.0 4 PMO, DAR3+

Referring to FIG. 15A, FIG. 15C, and FIG. 15E, in vivo exon skipping wasmeasured in wild type mice in the gastrocnemius (FIG. 15A), diaphragm(FIG. 15C) and heart muscle (FIG. 15E) using Tagman qPCR. Referring toFIG. 15B, FIG. 15D, and FIG. 15F, the CD71 mAb-PMO conjugates producedmeasurable exon23 skipping in gastrocnemius (FIG. 15B), diaphragm (FIG.15D), and heart muscle (FIG. 15F) using nested PCR. The wild typeproduct had an expected size of 788 bp, and the skipped DMD A23 had asize of 575 bp. The intensity of the bands on the gel was quantitated bydensitometry, and the data are presented as the % of skipped productcompared to wild-type dystrophin.

Example 9. In Vivo Activity of CD71 mAb-PMO Conjugates Against MouseMSTN

The CD71 mAb-PMO conjugate targeting exon 2 of mouse myostatin(5′AGCCCATCTTCTCCTGGTCCTGGGAAGG) (SEQ ID NO: 46) was made andcharacterized as described in Example 3. The conjugates (DAR1/2 andDAR3+) were assessed for its ability to mediate exon skipping in vivo inwild type CD-1 mice using similar methods as described in Example 2. Inbrief, mice were dosed via intravenous (iv) injection with the mAb,vehicle control and indicated ASCs at the doses indicated as seen inTable 15.

TABLE 15 In vivo study design mAb-PMO PMO PMO:mAb Harvest mAb dose DoseRatio Time Group Test Article N (mg/kg) (mg/kg) (mol/mol) (WEEKS) 1 CD713 50 5 1.5 1 mAb-PMO, DAR1/2 2 CD71 3 50 5 1.5 2 mAb-PMO, DAR1/2 3 CD713 50 5 1.5 4 mAb-PMO, DAR1/2 4 CD71 3 50 10 3.0 1 mAb-PMO, DAR3+ 5 CD713 50 10 3.0 2 mAb-PMO, DAR3+ 6 CD71 3 50 10 3.0 4 mAb-PMO. DAR3+ 7CD71-scr, 3 50 5 1.5 2 DAR1/2 8 CD71-scr, 3 50 10 3.0 2 DAR3+ 9 Vehicle3 1 10 Vehicle 3 2 11 Vehicle 3 4

After 7, 14 or 28 days, diaphragm, heart and gastrocnemius muscletissues were harvested and snap-frozen in liquid nitrogen. RNAs wereisolated and reversed transcribed. PCR reactions were performed withforward primer (mMSTN-F1: 5′CCTGGAAACAGCTCCTAACATC (SEQ ID NO: 50) andreverse primer (mMSTN-R1: 5′CAGTCAAGCCCAAAGTCTCTC (SEQ ID NO: 51) (hotstart: 95° C. for 2 minutes, Denaturation at 95° C. for 45 seconds,Annealing of primers at 56° C. for 30 seconds, primer extension at 72°C. for 40 seconds for 35 cycles). PCR reactions were analyzed in a 100TAE agarose gel as seen in FIGS. 16A-16C. The CD71 mAb-PMO conjugatesproduced measurable exon2 skipping in mouse diaphragm (FIG. 16A), heart(FIG. 16B) and gastrocnemius (FIG. 16C) muscle tissues. The wild typeproduct had an expected size of 622 bp and the skipped MSTN Δ2 of 248bp.

Example 10. In Vitro Activity of ASGPR mAb-PMO Conjugates Against thePAH Gene

The ASGPR mAb-PMO (5′ATCCTCTTTGGTAACCTCACCTCAC (SEQ ID NO: 47) conjugatetargeting exon 11 of mouse PAH was made and characterized as describedin Example 3. The conjugate was assessed for its ability to mediate exon11 skipping in the mouse PAH gene in vitro in primary mouse hepatocytesusing PCR (forward primer 5′-CTAGTGCCCTTGTTTTCAGA-3′ (SEQ ID NO: 52) andreverse primer 5′-AGGATCTACCACTGATGGGT-3′) (SEQ ID NO: 53). In brief,the potency of ASGPR mAb-PAH PMO conjugate was compared to ASGPRmAb-scramble PMO at multiple concentrations, with the relevant vehiclecontrol. RNAiMAX was also used to transfect the conjugates as positivecontrols. PCR reactions were analyzed in a 1% TAE agarose gel as seen inFIG. 17. As seen from the gel in FIG. 17, the ASGPR mAb-PMO conjugateproduced measurable exon11 skipping comparable to the RNAiMAXtransfected controls. The wild type product had an expected size of 703bp and the skipped PAH Δ11 of 569 bp.

Example 11. In Vivo Activity of ASGPR mAb-PMO Conjugates

The ASGPR mAb-PMO (5′ATCCTCTTTGGTAACCTCACCTCAC) (SEQ ID NO: 47)conjugate targeting exon 11 of mouse PAH was made and characterized asdescribed in Example 3. The conjugate (DAR1/2 and DAR3+) was assessedfor its ability to mediate exon skipping in vivo in wild type CD-1 miceusing methods as described in Example 2. In brief, mice were dosed viaintravenous (iv) injection with the mAb, vehicle control and indicatedASCs at the doses indicated as seen in Table 16.

TABLE 16 In vivo study design mAb-ASO PMO PMO:mAb Harvest mAb dose DoseRatio Time Group Test Article N (mg/kg) (mg/kg) (mol/mol) (Weeks) 1ASGPR 3 50 5 1.5 1 mAb-PMO, DAR1/2 2 ASGPR 3 50 5 1.5 2 mAb-PMO, DAR1/23 ASGPR 3 50 5 1.5 4 mAb-PMO, DAR1/2 4 ASGPR 2 50 10 3.0 1 mAb-PMO,DAR3+ 5 ASGPR 2 50 10 3.0 2 mAb-PMO, DAR3+ 6 ASGPR 2 50 10 3.0 4mAb-PMO, DAR3+ 7 ASGPR-Scr, 3 50 1.5 2 DAR1/2 8 ASGPR-Scr, 3 50 3.0 2DAR3+ 9 Vehicle 3 1 10 Vehicle 3 2 11 Vehicle 3 4

RNAs were isolated from harvested liver tissues and reverse transcribed.PCR reactions using forward primer 5′-CTAGTGCCCTTGTTTTCAGA-3′ (SEQ IDNO: 52) and reverse primer 5′-AGGATCTACCACTGATGGGT-3′ (SEQ ID NO: 53)were analyzed in a 1% TAE agarose gel as seen in FIG. 18. As can be seenfrom the gel in FIG. 18, the ASGPR mAb-PMO conjugates producedmeasurable exon11 skipping in mouse livers up to two weeks. The wildtype product had an expected size of 703 bp and the skipped PAH Δ11 of569 bp.

Example 12. Sequences

Table 17 illustrates exemplary target sequences to induce insertion,deletion, duplications, or alteration in the DMD gene using compositionsand methods as described herein. Table 18 illustrates exemplarynucleotide sequences to induce an insertion, deletion, duplication, oralteration in the DMD gene using compositions and methods as describedherein. Table 19 and Table 20 illustrate exemplary target sequences inseveral genes for inducing an insertion, deletion, duplications, oralteration in the gene. Table 21 illustrates exemplary sequences,including sequences in the DMD gene to induce an insertion, deletion,duplication, or alteration in the gene using compositions and methods asdescribed herein.

TABLE 17 Target SEQ Exon Antisense Sequence ID NO. 195′GCCUGAGCUGAUCUGCUGGCAUCUUGCAGU 54 U 3′ 19 or5′GCAGAAUUCGAUCCACCGGCUGUUCAAGCCUG 55 20 AGCUGAUCUGCUCGCAUCUUGCAGU 3′ 205′CAGCAGUAGUUGUCAUCUGCUC 3′ 56 21 5′CACAAAGUCUGCAUCCAGGAACAUGGGUC 3′ 5722 5′CUGCAAUUCCCCGAGUCUCUGC 3′ 58 51 5′CUCAUACCUUCUGCUUGAUGAUC 3′ 59 525′UCCAACUGGGGACGCCUCUGUUCCAAAUCC 3′ 60

TABLE 18 Target SEQ Gene Location Nucleotide Sequence (5′-3′) ID NO. DMDH8A(-06+18) GAUAGGUGGUAUCAACAUCUGUAA 61 DMD H8A(-03+18)GAUAGGUGGUAUCAACAUCUG 62 DMD H8A(-07+18) GAUAGGUGGUAUCAACAUCUGUAAG 63DMD H8A(-06+14) GGUGGUAUCAACAUCUGUAA 64 DMD H8A(-10+10)GUAUCAACAUCUGUAAGCAC 65 DMD H7A(+45+67) UGCAUGUUCCAGUCGUUGUGUGG 66 DMDH7A(+02+26) CACUAUUCCAGUCAAAUAGGUCUGG 67 DMD H7D(+15-10)AUUUACCAACCUUCAGGAUCGAGUA 68 DMD H7A(-18+03) GGCCUAAAACACAUACACAUA 69DMD C6A(-10+10) CAUUUUUGACCUACAUGUGG 70 DMD C6A(-14+06)UUUGACCUACAUGUGGAAAG 71 DMD C6A(-14+12) UACAUUUUUGACCUACAUGUGGAAAG 72DMD C6A(-13+09) AUUUUUGACCUACAUGGGAAAG 73 DMD CH6A(+69+91)UACGAGUUGAUUGUCGGACCCAG 74 DMD C6D(+12-13) GUGGUCUCCUUACCUAUGACUGUGG 75DMD C6D(+06-11) GGUCUCCUUACCUAUGA 76 DMD H6D(+04-21)UGUCUCAGUAAUCUUCUUACCUAU 77 DMD H6D(+18-04) UCUUACCUAUGACUAUGGAUGAGA 78DMD H4A(+13+32) GCAUGAACUCUUGUGGAUCC 79 DMD H4D(+04-16)CCAGGGUACUACUUACAUUA 80 DMD H4D(-24-44) AUCGUGUGUCACAGCAUCCAG 81 DMDH4A(+11+40) UGUUCAGGGCAUGAACUCUUGUGGAUCCUU 82 DMD H3A(+30+60)UAGGAGGCGCCUCCCAUCCUGUAGGUCACUG 83 DMD H3A(+35+65)AGGUCUAGGAGGCGCCUCCCAUCCUGUAGGU 84 DMD H3A(+30+54)GCGCCUCCCAUCCUGUAGGUCACUG 85 DMD H3D(+46-21) CUUCGAGGAGGUCUAGGAGGCGCCUC86 DMD H3A(+30+50) CUCCCAUCCUGUAGGUCACUG 87 DMD H3D(+19-03)UACCAGUUUUUGCCCUGUCAGG 88 DMD H3A(-06+20) UCAAUAUGCUGCUUCCCAAACUGAAA 89DMD H3A(+37+61) CUAGGAGGCGCCUCCCAUCCUGUAG 90 DMD H5A(+20+50)UUAUGAUUUCCAUCUACGAUGUCAGUACUUC 91 DMD H5D(+25-05)CUUACCUGCCAGUGGAGGAUUAUAUUCCAAA 92 DMD H5D(+10-15)CAUCAGGAUUCUUACCUGCCAGUGG 93 DMD H5A(+10+34) CGAUGUCAGUACUUCCAAUAUUCAC94 DMD H5D(-04-21) ACCAUUCAUCAGGAUUCU 95 DMD H5D(+16-02)ACCUGCCAGUGGAGGAUU 96 DMD H5A(-07+20) CCAAUAUUCACUAAAUCAACCUGUUAA 97 DMDH5D(+18-12) CAGGAUUGUUACCUGCCAGUGGAGGAUUAU 98 DMD H5A(+05+35)ACGAUGUCAGUACUUCCAAUAUUCACUAAAU 99 DMD H5A(+15+45)AUUUCCAUCUACGAUGUCAGUACUUCCAAUA 100 DMD H10A(-05+16)CAGGAGCUUCCAAAUGCUGCA 101 DMD H10A(-05+24) CUUGUCUUCAGGAGCUUCCAAAUGCUGCA102 DMD H10A(+98+119) UCCUCAGCAGAAAGAAGCCACG 103 DMD H10A(+130+149)UUAGAAAUCUCUCCUUGUGC 104 DMD H10A(-33-14) UAAAUUGGGUGUUACACAAU 105 DMDH11D(+26+49) CCCUGAGGCAUUCCCAUCUUGAAU 106 DMD H11D(+11-09)AGGACUUACUUGCUUUGUUU 107 DMD H11A(+118+140) CUUGAAUUUAGGAGAUUCAUCUG 108DMD H11A(+75+97) CAUCUUCUGAUAAUUUUCCUGUU 109 DMD H12A(+52+75)UCUUCUGUUUUUGUUAGCCAGUCA 110 DMD H12A(-10+10) UCUAUGUAAACUGAAAAUUU 111DMD H12A(+11+30) UUCUGGAGAUCCAUUAAAAC 112 DMD H13A(+77+100)CAGCAGUUGCGUGAUCUCCACUAG 113 DMD H13A(+55+75) UUCAUCAACUACCACCACCAU 114DMD H13D(+06-19) CUAAGCAAAAUAAUCUGACCUUAAG 115 DMD H14A(+37+64)CUUGUAAAAGAACCCAGCGGUCUUCUGU 116 DMD H14A(+14+35) CAUCUACAGAUGUUUGCCCAUC117 DMD H14A(+51+73) GAAGGAUGUCUUGUAAAAGAACC 118 DMD H14D(-02+18)ACCUGUUCUUCAGUAAGACG 119 DMD H14D(+14-10) CAUGACACACCUGUUCUUCAGUAA 120DMD H14A(+61+80) CAUUUGAGAAGGAUGUCUUG 121 DMD H14A(-12+12)AUCUCCCAAUACCUGGAGAAGAGA 122 DMD H15A(-12+19)GCCAUGCACUAAAAAGGCACUGCAAGACAUU 123 DMD H15A(+48+71)UCUUUAAAGCCAGUUGUGUGAAUC 124 DMD H15A(+08+28) UUUCUGAAAGCCAUGCACUAA 125DMD H15D(+17-08) GUACAUACGGCCAGUUUUUGAAGAC 126 DMD H16A(-12+19)CUAGAUCCGCUUUUAAAACCUGUUAAAACAA 127 DMD H16A(-06+25)UCUUUUCUAGAUCCGCUUUUAAAACCUGUUA 128 DMD H16A(-06+19)CUAGAUCCGCUUUUAAAACCUGUUA 129 DMD H16A(+87+109) CCGUCUUCUGGGUCACUGACUUA130 DMD H16A(-07+19) CUAGAUCCGCUUUUAAAACCUGUUAA 131 DMD H16A(-07+13)CCGCUUUUAAAACCUGUUAA 132 DMD H16A(+12+37) UGGAUUGCUUUUUCUUUUCUAGAUCC 133DMD H16A(+92+116) CAUGCUUCCGUCUUCUGGGUCACUG 134 DMD H16A(+45+67)GAUCUUGUUUGAGUGAAUACAGU 135 DMD H16A(+105+126) GUUAUCCAGCCAUGCUUCCGUC136 DMD H16D(+05-20) UGAUAAUUGGUAUCACUAACCUGUG 137 DMD H16D(+12-11)GUAUCACUAACCUGUGCUGUAC 138 DMD H19A(+35+53) CUGCUGGCAUCUUGCAGUU 139 DMDH19A(+35+65) GCCUGAGCUGAUCUGCUGGCAUCUUGCAGUU 140 DMD H20A(+44+71)CUGGCAGAAUUCGAUCCACCGGCUGUUC 141 DMD H20A(+147+168)CAGCAGUAGUUGUCAUCUGCUC 142 DMD H20A(+185+203) UGAUGGGGUGGUGGGUUGG 143DMD H20A(-08+17) AUCUGCAUUAACACCCUCUAGAAAG 144 DMD H20A(+30+53)CCGGCUGUUCAGUUGUUCUGAGGC 145 DMD H20A(-11+17)AUCUGCAUUAACACCCUCUAGAAAGAAA 146 DMD H20D(+08-20)GAAGGAGAAGAGAUUCUUACCUUACAAA 147 DMD H20A(+44+63) AUUCGAUCCACCGGCUGUUC148 DMD H20A(+149+168 CAGCAGUAGUUGUCAUCUGC 149 DMD H21A(-06+16)GCCGGUUGACUUCAUCCUGUGC 150 DMD H21A(+85+106) CUGCAUCCAGGAACAUGGGUCC 151DMD H21A(+85+108) GUCUGCAUCCAGGAACAUGGGUC 152 DMD H21A(+08+31)GUUGAAGAUCUGAUAGCCGGUUGA 153 DMD H21D(+18-07) UACUUACUGUCUGUAGCUCUUUCU154 DMD H22A(+22+45) CACUCAUGGUCUCCUGAUAGCGCA 155 DMD H22A(+125+106)CUGCAAUUCCCCGAGUCUCUGC 156 DMD H22A(+47+69) ACUGCUGGACCCAUGUCCUGAUG 157DMD H22A(+80+101) CUAAGUUGAGGUAUGGAGAGU 158 DMD H22D(+13-11)UAUUCACAGACCUGCAAUUCCCC 159 DMD H23A(+34+59) ACAGUGGUGCUGAGAUAGUAUAGGCC160 DMD H23A(+18+39) UAGGCCACUUUGUUGCUCUUGC 161 DMD H23A(+72+90)UUCAGAGGGCGCUUUCUUC 162 DMD H24A(+48+70) GGGCAGGCCAUUCCUCCUUCAGA 163 DMDH24A(-02+22) UCUUCAGGGUUUGUAUGUGAUUCU 164 DMD H25A(+9+36)CUGGGCUGAAUUGUCUGAAUAUCACUG 165 DMD H25A(+131+156)CUGUUGGCACAUGUGAUCCCACUGAG 166 DMD H25D(+16-08) GUCUAUACCUGUUGGCACAUGUGA167 DMD H26A(+132+156) UGCUUUCUGUAAUUCAUCUGGAGUU 168 DMD H26A(-07+19)CCUCCUUUCUGGCAUAGACCUUCCAC 169 DMD H26A(+68+92)UGUGUCAUCCAUUCGUGCAUCUCUG 170 DMD H27A(+82+106)UUAAGGCCUCUUGUGCUACAGGUGG 171 DMD H27A(-4+19) GGGGCUCUUCUUUAGCUCUCUGA172 DMD H27D(+19-03) GACUUCCAAAGUCUUGCAUUUC 173 DMD H28A(-05+19)GCCAACAUGCCCAAACUUCCUAAG 174 DMD H28A(+99+124)CAGAGAUUUCCUCAGCUCCGCCAGGA 175 DMD H28D(+16-05) CUUACAUCUAGCACCUCAGAG176 DMD H29A(+57+81) UCCGCCAUCUGUUAGGGUCUGUGCC 177 DMD H29A(+18+42)AUUUGGGUUAUCCUCUGAAUGUCGC 178 DMD H29D(+17-05) CAUACCUCUUCAUGUAGUUCCC179 DMD H30A(+122+147) CAUUUGAGCUGCGUCCACCUUGUCUG 180 DMD H30A(+25+50)UCCUGGGCAGACUGGAUGCUCUGUUC 181 DMD H30D(+19-04) UUGCCUGGGCUUCCUGAGGCAUU182 DMD H31D(+06-18) UUCUGAAAUAACAUAUACCUGUGC 183 DMD H31D(+03-22)UAGUUUCUGAAAUAACAUAUACCUG 184 DMD H31A(+05+25) GACUUGUCAAAUCAGAUUGGA 185DMD H31D(+04-20) GUUUCUGAAAUAACAUAUACCUGU 186 DMD H32D(+04-16)CACCAGAAAUACAUACCACA 187 DMD H32A(+151+170) CAAUGAUUUAGCUGUGACUG 188 DMDH32A(+10+32) CGAAACUUCAUGGAGACAUCUUG 189 DMD H32A(+49+73)CUUGUAGACGCUGCUCAAAAUUGGC 190 DMD H33D(+09-11) CAUGCACACACCUUUGCUCC 191DMD H33A(+53+76) UCUGUACAAUCUGACGUCCAGUCU 192 DMD H33A(+30+56)GUCUUUAUCACCAUUUCCACUUCAGAC 193 DMD H33A(+64+88)CCGUCUGCUUUUUCUGUACAAUCUG 194 DMD H34A(+83+104) UCCAUAUCUGUAGCUGCCAGCC195 DMD H34A(+143+165) CCAGGCAACUUCAGAAUCCAAAU 196 DMD H34A(-20+10)UUUCUGUUACCUGAAAAGAAUUAUAAUGAA 197 DMD H34A(+46+70)CAUUCAUUUCCUUUCGCAUCUUACG 198 DMD H34A(+95+120)UGAUCUCUUUGUCAAUUCCAUAUCUG 199 DMD H34D(+10-20)UUCAGUGAUAUAGGUUUUACCUUUCCCCAG 200 DMD H34A(+72+96) CUG UAG CUG CCA GCCAUU CUG UCA AG 201 DMD H35A(+141+161) UCU UCU GCU CGG GAG GUG ACA 202DMD H35A(+116+135) CCA GUU ACU AUU CAG AAG AC 203 DMD H35A(+24+43) UCUUCA GGU GCA CCU UCU GU 204 DMD H36A(+26+50) UGUGAUGUGGUCCACAUUCUGGUCA205 DMD H36A(-02+18) CCAUGUGUUUCUGGUAUUCC 206 DMD H37A(+26+50)CGUGUAGAGUCCACCUUUGGGCGUA 207 DMD H37A(+82+105) UACUAAUUUCCUGCAGUGGUCACC208 DMD H37A(+134+157) UUCUGUGUGAAAUGGCUGCAAAUC 209 DMD H38A(-01+19)CCUUCAAAGGAAUGGAGGCC 210 DMD H38A(+59+83) UGCUGAAUUUCAGCCUCCAGUGGUU 211DMD H38A(+88+112) UGAAGUCUUCCUCUUUCAGAUUCAC 212 DMD H39A(+62+85)CUGGCUUUCUCUCAUCUGUGAUUC 213 DMD H39A(+39+58) GUUGUAAGUUGUCUCCUCUU 214DMD H39A(+102+121) UUGUCUGUAACAGCUGCUGU 215 DMD H39D(+10-10)GCUCUAAUACCUUGAGAGCA 216 DMD H40A(-05+17) CUUUGAGACCUCAAAUCCUGUU 217 DMDH40A(+129+153) CUUUAUUUUCCUUUCAUCUCUGGGC 218 DMD H42A(-04+23)AUCGUUUCUUCACGGACAGUGUGCUGG 219 DMD H42A(+86+109)GGGCUUGUGAGACAUGAGUGAUUU 220 DMD H42D(+19-02) ACCUUCAGAGGACUCCUCUUGC 221DMD H43D(+10-15) UAUGUGUUACCUACCCUUGUCGGUC 222 DMD H43A(+101+120)GGAGAGAGCUUCCUGUAGCU 223 DMD H43A(+78+100) UCACCCUUUCCACAGGCGUUGCA 224DMD H44A(+85+104) UUUGUGUCUUUCUGAGAAAC 225 DMD H44D(+10-10)AAAGACUUACCUUAAGAUAC 226 DMD H44A(-06+14) AUCUGUCAAAUCGCCUGCAG 227 DMDH46D(+16-04) UUACCUUGACUUGCUCAAGC 228 DMD H46A(+90+109)UCCAGGUUCAAGUGGGAUAC 229 DMD H47A(+76+100) GCUCUUCUGGGCUUAUGGGAGCACU 230DMD H47D(+25-02) ACCUUUAUCCACUGGAGAUUUGUCUGC 231 DMD H47A(-9+12)UUCCACCAGUAACUGAAACAG 232 DMD H50A(+02+30) CCACUCAGAGCUCAGAUCUUCUAACUUCC233 DMD H50A(+07+33) CUUCCACUCAGAGCUCAGAUCUUCUAA 234 DMD H50D(+07-18)GGGAUCCAGUAUACUUACAGGCUCC 235 DMD H51A(-01+25)ACCAGAGUAACAGUCUGAGUAGGAGC 236 DMD H51D(+16-07) CUCAUACCUUCUGCUUGAUGAUC237 DMD H51A(+111+134) UUCUGUCCAAGCCCGGUUGAAAUC 238 DMD H51A(+61+90)ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG 239 DMD H51A(+66+90)ACAUCAAGGAAGAUGGCAUUUCUAG 240 DMD H51A(+66+95)CUCCAACAUCAAGGAAGAUGGCAUUUCUAG 241 DMD H51D(+08-17)AUCAUUUUUUCUCAUACCUUCUGCU 242 DMD H51A/D(+08-17)AUCAUUUUUUCUCAUACCUUCUGCUAG 243 &(-15+) GAGCUAAAA DMD H51A(+175+195)CACCCACCAUCACCCUCUGUG 245 DMD H51A(+199+220) AUCAUCUCGUUGAUAUCCUCAA 246DMD H52A(-07+14) UCCUGCAUUGUUGCCUGUAAG 247 DMD H52A(+12+41)UCCAACUGGGGACGCCUCUGUUCCAAAUCC 248 DMD H52A(+17+37)ACUGGGGACGCCUCUGUUCCA 249 DMD H52A(+93+112) CCGUAAUGAUUGUUCUAGCC 250 DMDH52D(+05-15) UGUUAAAAAACUUACUUCGA 251 DMD H53A(+45+69)CAUUCAACUGUUGCCUCCGGUUCUG 252 DMD H53A(+39+62) CUGUUGCCUCCGGUUCUGAAGGUG253 DMD H53A(+39+69) CAUUCAACUGUUGCCUCCGGUUCUGAAGGUG 254 DMDH53D(+14-07) UACUAACCUUGGUUUCUGUGA 255 DMD H53A(+23+47)CUGAAGGUGUUCUUGUACUUCAUCC 256 DMD H53A(+150+176)UGUAUAGGGACCCUCCUUCCAUGACUC 257 DMD H53D(+20-05)CUAACCUUGGUUUCUGUGAUUUUCU 258 DMD H53D(+09-18)GGUAUCUUUGAUACUAACCUUGGUUUC 259 DMD H53A(-12+10) AUUCUUUCAACUAGAAUAAAAG260 DMD H53A(-07+18) GAUUCUGAAUUCUUUCAACUAGAAU 261 DMD H53A(+07+26)AUCCCACUGAUUCUGAAUUC 262 DMD H53A(+124+145) UUGGCUCUGGCCUGUCCUAAGA 263DMD H46A(+86+115) CUCUUUUCCAGGUUCAAGUGGGAUACUAGC 264 DMD H46A(+107+137)CAAGCUUUUCUUUUAGUUGCUGCUCUUUUCC 265 DMD H46A(-10+20)UAUUCUUUUGUUCUUCUAGCCUGGAGAAAG 266 DMD H46A(+50+77)CUGCUUCCUCCAACCAUAAAACAAAUUC 267 DMD H45A(-06+20)CCAAUGCCAUCCUGGAGUUCCUGUAA 268 DMD H45A(+91+110) UCCUGUAGAAUACUGGCAUC269 DMD H45A(+125+151) UGCAGACCUCCUGCCACCGCAGAUUCA 270 DMD H45D(+16-04)CUACCUCUUUUUUCUGUCUG 271 DMD H45A(+71+90) UGUUUUUGAGGAUUGCUGAA 272 * Thefirst letter designates the species (e.g. H: human, M: murine, C:canine). “#”designates target DMD exon number. “A/D”indicates acceptoror donor splice site at the beginning and end of the exon, respectively.(x y) represents the annealing coordinates where “-”or “+”indicateintronic or exonic sequences respectively.

TABLE 19 Nucleotide Sequence SEQ Gene (5′-3′) ID NO. Bcl-xTGGTTCTTACCCAGCCGCCG 273 β-globin 623 GTTATTCTTTAGAATGGTGC 274 β-globin654 TGCTATTACCTTAACCCAGA 275 c-myc CTGTGCTTACCGGGTTTTCC 276 ACCTCCCc-myc ATCGTCGTGACTGTCTGTTG 277 GAGGG c-myc GCTCACGTTGAGGGGCATCG 278c-myc ACGTTGAGGGGCATCGTCGC 279 c-myc GGGGCAUCGUCGUGACUGU/ 280CUGUUGGAGGG c-myc CGUCGUGACUGUCUGUUGGA 281 GG c-myc CGTCGTGACTGTCTGTTGGA282 GG c-myc GGCAUCGUCGCGGGAGGCUG 283 CUGGAGCG c-mycCCGCGACAUAGGACGGAGAG 284 CAGAGCCC c-myc ACTGTGAGGGCGATCGCTGC 285 c-mycACGATGAGTGGCATAGTCGC 286 c-myc GGCATCGTCGCGGGAGGCTG 287 c-mycGGGCATCGTCGCGGGAGGCT 288 c-myc GGGGCATCGTCGCGGGAGGC 289 c-mycAGGGGCATCGTCGCGGGAGG 290 c-myc GAGGGGCATCGTCGCGGGAG 291 c-mycTGAGGGGCATCGTCGCGGGA 292 c-myc TTGAGGGGCATCGTCGCGGG 293 c-mycGTTGAGGGGCATCGTCGCGG 294 c-myc CGTTGAGGGGCATCGTCGCG 295 c-mycACGTTGAGGGGCATCGTCGC 296 c-myc AACGTTGAGGGGCATCGTCG 297 c-mycTAACGTTGAGGGGCATCGTC 298 c-myc CTAACGTTGAGGGGCATCGT 299 c-mycGCTAACGTTGAGGGGCATCG 300 c-myc AGCTAACGTTGAGGGGCATC 301 c-mycAAGCTAACGTTGAGGGGCAT 302 c-myc GAAGCTAACGTTGAGGGGCA 303 BCL-2 (rat)CTCCGCAATGCTGAAAGGTG 304 PCNA-1 (rat) GGCGUGCCUCAAACAUGGUG 305 GCGG

TABLE 20 Target SEQ Gene Location Nucleotide Sequence (5′-3′) ID NO. Ratc-myc 2553-79 CTGTGCTTACCGGGTTTTCCACCTCCC 306 Rat c-myc 4140-64ATCGTCGTGACTGTCTGTTGGAGGG 307 Rat c-myc 4161-80 GCTCACGTTGAGGGGCATCG 308Rat CYP3A2 1155-74 GGTCACTCACCGGTAGAGAA 309 Rat CYP3A2 1526-45GGGTTCCAAGTCTATAAAGG 310 Human 31-44 TGTGTCTTTTCCAG 311 androgenreceptor exon 2 Human 45-67 TTTGGAGACTGCCAGGGACCATG 312 androgenreceptor exon 2 Human 48-67 CATGGTCCCTGGCAGTCTCC 313 androgen receptorexon 2 Human 45-80 TCAATGGGCAAAACATGGTCCCTGGCAGTCTCCAAA 314 androgenreceptor exon 2 Human 28-43 TTTGTGTTCTCCCAG 315 androgen receptor exon 3Human 44-66 GGAAACAGAAGTACCTGTGCGCC 316 androgen receptor exon 3 Human49-66 GGCGCACAGGTACTTCTG 317 androgen receptor exon 3 Human 44-79AATCATTTCTGCTGGCGCACAGGTACTTCTGTTTCC 318 androgen receptor exon 3 HumanHCG-β 1321-38 CCCCTGCAGCACGCGGGT 319 subunit Human HCG-β 1321-57GAGGCAGGGCCGGCAGGACCCCCTGCAGCACGCGGGT 320 subunit Human c-myc 4506-25GGCATCGTCGCGGGAGGCTG 321 Human c-myc 4507-26 GGGCATCGTCGCGGGAGGCT 322Human c-myc 4508-27 GGGGCATCGTCGCGGGAGGC 323 Human c-myc 4509-28AGGGGCATCGTCGCGGGAGG 324 Human c-myc 4510-29 GAGGGGCATCGTCGCGGGAG 325Human c-myc 4511-30 TGAGGGGCATCGTCGCGGGA 326 Human c-myc 4512-31TTGAGGGGCATCGTCGCGGG 327 Human c-myc 4513-32 GTTGAGGGGCATCGTCGCGG 328Human c-myc 4514-33 CGTTGAGGGGCATCGTCGCG 329 Human c-myc 4515-34ACGTTGAGGGGCATCGTCGC 330 Human c-myc 4516-35 AACGTTGAGGGGCATCGTCG 331Human c-myc 4517-36 TAACGTTGAGGGGCATCGTC 332 Human c-myc 4518-37CTAACGTTGAGGGGCATCGT 333 Human c-myc 4519-38 GCTAACGTTGAGGGGCATCG 334Human c-myc 4520-39 AGCTAACGTTGAGGGGCATC 335 Human c-myc 4521-40AAGCTAACGTTGAGGGGCAT 336 Human c-myc 4522-41 GAAGCTAACGTTGAGGGGCA 337Human c-myc 6656-75 TCCTCATCTTCTTGTTCCTC 338 Human c-myc 6656-91AACAACATCGATTTCTTCCTCATCTTCTTGTTCCTC 339 Human p53 11691-708CCCGGAAGGCAGTCTGGC 340 Human p53 11689-724TCCTCCATGGCAGTGACCCGGAAGGCAGTCTGGCTG 341 Human abl (ds 376-94CTACTGGCCGCTGAAGGGC 342 of bcr-abl fusion point) Human abl (ds 374-409GCTCAAAGTCAGATGCTACTGGCCGCTGAAGGGCTT 343 of bcr-abl fusion point) HW-1rev 5517-43 TCGTCGGTCTCTCCGCTTCTTCTTGCC 344 HW-1 rev 7885-7904CTCTGGTGGTGGGTAAGGGT 345 HW-1 rev 7885-7921CGGGTCTGTCGGGTTCCCTCTGGTGGTGGGTAAGGGT 346 Rat c-myc 4140-69GGGGCAUCGUCGUGACUGUCUGUUGGAGGG 347 Rat c-myc 4141-62CGUCGUGACUGUCUGUUGGAGG 348 Rat c-myc 4141-62 CGTCGTGACTGTCTGTTGGAGG 349Human c-myc 4498-4505 GGCAUCGUCGCGGGAGGCUG/CUGGAGCG 350 Rat c-myc4364-91 CCGCGACAUAGGACGGAGAGCAGAGCCC 351

TABLE 21 SEQ Target Nucleotide Sequence (5′-3′) ID NO.Hu.DMD.Exon44.25.001 CTGCAGGTAAAAGCATATGGATCAA 352 Hu.DMD.Exon44.25.002ATCGCCTGCAGGTAAAAGCATATGG 353 Hu.DMD.Exon44.25.003GTCAAATCGCCTGCAGGTAAAAGCA 354 Hu.DMD.Exon44.25.004GATCTGTCAAATCGCCTGCAGGTAA 355 Hu.DMD.Exon44.25.005CAACAGATCTGTCAAATCGCCTGCA 356 Hu.DMD.Exon44.25.006TTTCTCAACAGATCTGTCAAATCGC 357 Hu.DMD.Exon44.25.007CCATTTCTCAACAGATCTGTCAAAT 358 Hu.DMD.Exon44.25.008ATAATGAAAACGCCGCCATTTCTCA 359 Hu.DMD.Exon44.25.009AAATATCTTTATATCATAATGAAAA 360 Hu.DMD.Exon44.25.010TGTTAGCCACTGATTAAATATCTTT 361 Hu.DMD.Exon44.25.011AAACTGTTCAGCTTCTGTTAGCCAC 362 Hu.DMD.Exon44.25.012TTGTGTCTTTCTGAGAAACTGTTCA 363 Hu.DMD.Exon44.25.013CCAATTCTCAGGAATTTGTGTCTTT 364 Hu.DMD.Exon44.25.014GTATTTAGCATGTTCCCAATTCTCA 365 Hu.DMD.Exon44.25.015CTTAAGATACCATTTGTATTTAGCA 366 Hu.DMD.Exon44.25.016CTTACCTTAAGATACCATTTGTATT 367 Hu.DMD.Exon44.25.017AAAGACTTACCTTAAGATACCATTT 368 Hu.DMD.Exon44.25.018AAATCAAAGACTTACCTTAAGATAC 369 Hu.DMD.Exon44.25.019AAAACAAATCAAAGACTTACCTTAA 370 Hu.DMD.Exon44.25.020TCGAAAAAACAAATCAAAGACTTAC 371 Hu.DMD.Exon45.25.001CTGTAAGATACCAAAAAGGCAAAAC 372 Hu.DMD.Exon45.25.002CCTGTAAGATACCAAAAAGGCAAAA 373 Hu.DMD.Exon45.25.002.2AGTTCCTGTAAGATACCAAAAAGGC 374 Hu.DMD.Exon45.25.003GAGTTCCTGTAAGATACCAAAAAGG 375 Hu.DMD.Exon45.25.003.2CCTGGAGTTCCTGTAAGATACCAAA 376 Hu.DMD.Exon45.25.004TCCTGGAGTTCCTGTAAGATACCAA 377 Hu.DMD.Exon45.25.004.2GCCATCCTGGAGTTCCTGTAAGATA 378 Hu.DMD.Exon45.25.005TGCCATCCTGGAGTTCCTGTAAGAT 379 Hu.DMD.Exon45.25.005.2CCAATGCCATCCTGGAGTTCCTGTA 380 Hu.DMD.Exon45.25.006CCCAATGCCATCCTGGAGTTCCTGT 381 Hu.DMD.Exon45.25.006.2GCTGCCCAATGCCATCCTGGAGTTC 382 Hu.DMD.Exon45.25.007CGCTGCCCAATGCCATCCTGGAGTT 383 Hu.DMD.Exon45.25.008AACAGTTTGCCGCTGCCCAATGCCA 384 Hu.DMD.Exon45.25.008.2CTGACAACAGTTTGCCGCTGCCCAA 385 Hu.DMD.Exon45.25.009GTTGCATTCAATGTTCTGACAACAG 386 Hu.DMD.Exon45.25.010GCTGAATTATTTCTTCCCCAGTTGC 387 Hu.DMD.Exon45.25.010.2ATTATTTCTTCCCCAGTTGCATTCA 388 Hu.DMD.Exon45.25.011GGCATCTGTTTTTGAGGATTGCTGA 389 Hu.DMD.Exon45.25.011.2TTTGAGGATTGCTGAATTATTTCTT 390 Hu.DMD.Exon45.25.012AATTTTTCCTGTAGAATACTGGCAT 391 Hu.DMD.Exon45.25.012.2ATACTGGCATCTGTTTTTGAGGATT 392 Hu.DMD.Exon45.25.013ACCGCAGATTCAGGCTTCCCAATTT 393 Hu.DMD.Exon45.25.013.2AATTTTTCCTGTAGAATACTGGCAT 394 Hu.DMD.Exon45.25.014CTGTTTGCAGACCTCCTGCCACCGC 395 Hu.DMD.Exon45.25.014.2AGATTCAGGCTTCCCAATTTTTCCT 396 Hu.DMD.Exon45.25.015CTCTTTTTTCTGTCTGACAGCTGTT 397 Hu.DMD.Exon45.25.015.2ACCTCCTGCCACCGCAGATTCAGGC 398 Hu.DMD.Exon45.25.016CCTACCTCTTTTTTCTGTCTGACAG 399 Hu.DMD.Exon45.25.016.2GACAGCTGTTTGCAGACCTCCTGCC 400 Hu.DMD.Exon45.25.017GTCGCCCTACCTCTTTTTTCTGTCT 401 Hu.DMD.Exon45.25.018GATCTGTCGCCCTACCTCTTTTTTC 402 Hu.DMD.Exon45.25.019TATTAGATCTGTCGCCCTACCTCTT 403 Hu.DMD.Exon45.25.020ATTCCTATTAGATCTGTCGCCCTAC 404 Hu.DMD.Exon45.20.001 AGATACCAAAAAGGCAAAAC405 Hu.DMD.Exon45.20.002 AAGATACCAAAAAGGCAAAA 406 Hu.DMD.Exon45.20.003CCTGTAAGATACCAAAAAGG 407 Hu.DMD.Exon45.20.004 GAGTTCCTGTAAGATACCAA 408Hu.DMD.Exon45.20.005 TCCTGGAGTTCCTGTAAGAT 409 Hu.DMD.Exon45.20.006TGCCATCCTGGAGTTCCTGT 410 Hu.DMD.Exon45.20.007 CCCAATGCCATCCTGGAGTT 411Hu.DMD.Exon45.20.008 CGCTGCCCAATGCCATCCTG 412 Hu.DMD.Exon45.20.009CTGACAACAGTTTGCCGCTG 413 Hu.DMD.Exon45.20.010 GTTGCATTCAATGTTCTGAC 414Hu.DMD.Exon45.20.011 ATTATTTCTTCCCCAGTTGC 415 Hu.DMD.Exon45.20.012TTTGAGGATTGCTGAATTAT 416 Hu.DMD.Exon45.20.013 ATACTGGCATCTGTTTTTGA 417Hu.DMD.Exon45.20.014 AATTTTTCCTGTAGAATACT 418 Hu.DMD.Exon45.20.015AGATTCAGGCTTCCCAATTT 419 Hu.DMD.Exon45.20.016 ACCTCCTGCCACCGCAGATT 420Hu.DMD.Exon45.20.017 GACAGCTGTTTGCAGACCTC 421 Hu.DMD.Exon45.20.018CTCTTTTTTCTGTCTGACAG 422 Hu.DMD.Exon45.20.019 CCTACCTCTTTTTTCTGTCT 423Hu.DMD.Exon45.20.020 GTCGCCCTACCTCTTTTTTC 424 Hu.DMD.Exon45.20.021GATCTGTCGCCCTACCTCTT 425 Hu.DMD.Exon45.20.022 TATTAGATCTGTCGCCCTAC 426Hu.DMD.Exon45.20.023 ATTCCTATTAGATCTGTCGC 427 Hu.DMD.Exon46.25.001GGGGGATTTGAGAAAATAAAATTAC 428 Hu.DMD.Exon46.25.002ATTTGAGAAAATAAAATTACCTTGA 429 Hu.DMD.Exon46.25.002.2CTAGCCTGGAGAAAGAAGAATAAAA 430 Hu.DMD.Exon46.25.003AGAAAATAAAATTACCTTGACTTGC 431 Hu.DMD.Exon46.25.003.2TTCTTCTAGCCTGGAGAAAGAAGAA 432 Hu.DMD.Exon46.25.004ATAAAATTACCTTGACTTGCTCAAG 433 Hu.DMD.Exon46.25.004.2TTTTGTTCTTCTAGCCTGGAGAAAG 434 Hu.DMD.Exon46.25.005ATTACCTTGACTTGCTCAAGCTTTT 435 Hu.DMD.Exon46.25.005.2TATTCTTTTGTTCTTCTAGCCTGGA 436 Hu.DMD.Exon46.25.006CTTGACTTGCTCAAGCTTTTCTTTT 437 Hu.DMD.Exon46.25.006.2CAAGATATTCTTTTGTTCTTCTAGC 438 Hu.DMD.Exon46.25.007CTTTTAGTTGCTGCTCTTTTCCAGG 439 Hu.DMD.Exon46.25.008CCAGGTTCAAGTGGGATACTAGCAA 440 Hu.DMD.Exon46.25.008.2ATCTCTTTGAAATTCTGACAAGATA 441 Hu.DMD.Exon46.25.009AGCAATGTTATCTGCTTCCTCCAAC 442 Hu.DMD.Exon46.25.009.2AACAAATTCATTTAAATCTCTTTGA 443 Hu.DMD.Exon46.25.010CCAACCATAAAACAAATTCATTTAA 444 Hu.DMD.Exon46.25.010.2TTCCTCCAACCATAAAACAAATTCA 445 Hu.DMD.Exon46.25.011TTTAAATCTCTTTGAAATTCTGACA 446 Hu.DMD.Exon46.25.012TGACAAGATATTCTTTTGTTCTTCT 447 Hu.DMD.Exon46.25.012.2TTCAAGTGGGATACTAGCAATGTTA 448 Hu.DMD.Exon46.25.013AGATATTCTTTTGTTCTTCTAGCCT 449 Hu.DMD.Exon46.25.013.2CTGCTCTTTTCCAGGTTCAAGTGGG 450 Hu.DMD.Exon46.25.014TTCTTTTGTTCTTCTAGCCTGGAGA 451 Hu.DMD.Exon46.25.014.2CTTTTCTTTTAGTTGCTGCTCTTTT 452 Hu.DMD.Exon46.25.015TTGTTCTTCTAGCCTGGAGAAAGAA 453 Hu.DMD.Exon46.25.016CTTCTAGCCTGGAGAAAGAAGAATA 454 Hu.DMD.Exon46.25.017AGCCTGGAGAAAGAAGAATAAAATT 455 Hu.DMD.Exon46.25.018CTGGAGAAAGAAGAATAAAATTGTT 456 Hu.DMD.Exon46.20.001 GAAAGAAGAATAAAATTGTT457 Hu.DMD.Exon46.20.002 GGAGAAAGAAGAATAAAATT 458 Hu.DMD.Exon46.20.003AGCCTGGAGAAAGAAGAATA 459 Hu.DMD.Exon46.20.004 CTTCTAGCCTGGAGAAAGAA 460Hu.DMD.Exon46.20.005 TTGTTCTTCTAGCCTGGAGA 461 Hu.DMD.Exon46.20.006TTCTTTTGTTCTTCTAGCCT 462 Hu.DMD.Exon46.20.007 TGACAAGATATTCTTTTGTT 463Hu.DMD.Exon46.20.008 ATCTCTTTGAAATTCTGACA 464 Hu.DMD.Exon46.20.009AACAAATTCATTTAAATCTC 465 Hu.DMD.Exon46.20.010 TTCCTCCAACCATAAAACAA 466Hu.DMD.Exon46.20.011 AGCAATGTTATCTGCTTCCT 467 Hu.DMD.Exon46.20.012TTCAAGTGGGATACTAGCAA 468 Hu.DMD.Exon46.20.013 CTGCTCTTTTCCAGGTTCAA 469Hu.DMD.Exon46.20.014 CTTTTCTTTTAGTTGCTGCT 470 Hu.DMD.Exon46.20.015CTTGACTTGCTCAAGCTTTT 471 Hu.DMD.Exon46.20.016 ATTACCTTGACTTGCTCAAG 472Hu.DMD.Exon46.20.017 ATAAAATTACCTTGACTTGC 473 Hu.DMD.Exon46.20.018AGAAAATAAAATTACCTTGA 474 Hu.DMD.Exon46.20.019 ATTTGAGAAAATAAAATTAC 475Hu.DMD.Exon46.20.020 GGGGGATTTGAGAAAATAAA 476 Hu.DMD.Exon47.25.001CTGAAACAGACAAATGCAACAACGT 477 Hu.DMD.Exon47.25.002AGTAACTGAAACAGACAAATGCAAC 478 Hu.DMD.Exon47.25.003CCACCAGTAACTGAAACAGACAAAT 479 Hu.DMD.Exon47.25.004CTCTTCCACCAGTAACTGAAACAGA 480 Hu.DMD.Exon47.25.005GGCAACTCTTCCACCAGTAACTGAA 481 Hu.DMD.Exon47.25.006GCAGGGGCAACTCTTCCACCAGTAA 482 Hu.DMD.Exon47.25.007CTGGCGCAGGGGCAACTCTTCCACC 483 Hu.DMD.Exon47.25.008TTTAATTGTTTGAGAATTCCCTGGC 484 Hu.DMD.Exon47.25.008.2TTGTTTGAGAATTCCCTGGCGCAGG 485 Hu.DMD.Exon47.25.009GCACGGGTCCTCCAGTTTCATTTAA 486 Hu.DMD.Exon47.25.009.2TCCAGTTTCATTTAATTGTTTGAGA 487 Hu.DMD.Exon47.25.010GCTTATGGGAGCACTTACAAGCACG 488 Hu.DMD.Exon47.25.010.2TACAAGCACGGGTCCTCCAGTTTCA 489 Hu.DMD.Exon47.25.011AGTTTATCTTGCTCTTCTGGGCTTA 490 Hu.DMD.Exon47.25.012TCTGCTTGAGCTTATTTTCAAGTTT 491 Hu.DMD.Exon47.25.012.2ATCTTGCTCTTCTGGGCTTATGGGA 492 Hu.DMD.Exon47.25.013CTTTATCCACTGGAGATTTGTCTGC 493 Hu.DMD.Exon47.25.013.2CTTATTTTCAAGTTTATCTTGCTCT 494 Hu.DMD.Exon47.25.014CTAACCTTTATCCACTGGAGATTTG 495 Hu.DMD.Exon47.25.014.2ATTTGTCTGCTTGAGCTTATTTTCA 496 Hu.DMD.Exon47.25.015AATGTCTAACCTTTATCCACTGGAG 497 Hu.DMD.Exon47.25.016TGGTTAATGTCTAACCTTTATCCAC 498 Hu.DMD.Exon47.25.017AGAGATGGTTAATGTCTAACCTTTA 499 Hu.DMD.Exon47.25.018ACGGAAGAGATGGTTAATGTCTAAC 500 Hu.DMD.Exon47.20.001 ACAGACAAATGCAACAACGT501 Hu.DMD.Exon47.20.002 CTGAAACAGACAAATGCAAC 502 Hu.DMD.Exon47.20.003AGTAACTGAAACAGACAAAT 503 Hu.DMD.Exon47.20.004 CCACCAGTAACTGAAACAGA 504Hu.DMD.Exon47.20.005 CTCTTCCACCAGTAACTGAA 505 Hu.DMD.Exon47.20.006GGCAACTCTTCCACCAGTAA 506 Hu.DMD.Exon47.20.007 CTGGCGCAGGGGCAACTCTT 507Hu.DMD.Exon47.20.008 TTGTTTGAGAATTCCCTGGC 508 Hu.DMD.Exon47.20.009TCCAGTTTCATTTAATTGTT 509 Hu.DMD.Exon47.20.010 TACAAGCACGGGTCCTCCAG 510Hu.DMD.Exon47.20.011 GCTTATGGGAGCACTTACAA 511 Hu.DMD.Exon47.20.012ATCTTGCTCTTCTGGGCTTA 512 Hu.DMD.Exon47.20.013 CTTATTTTCAAGTTTATCTT 513Hu.DMD.Exon47.20.014 ATTTGTCTGCTTGAGCTTAT 514 Hu.DMD.Exon47.20.015CTTTATCCACTGGAGATTTG 515 Hu.DMD.Exon47.20.016 CTAACCTTTATCCACTGGAG 516Hu.DMD.Exon47.20.017 AATGTCTAACCTTTATCCAC 517 Hu.DMD.Exon47.20.018TGGTTAATGTCTAACCTTTA 518 Hu.DMD.Exon47.20.019 AGAGATGGTTAATGTCTAAC 519Hu.DMD.Exon47.20.020 ACGGAAGAGATGGTTAATGT 520 Hu.DMD.Exon48.25.001CTGAAAGGAAAATACATTTTAAAAA 521 Hu.DMD.Exon48.25.002CCTGAAAGGAAAATACATTTTAAAA 522 Hu.DMD.Exon48.25.002.2GAAACCTGAAAGGAAAATACATTTT 523 Hu.DMD.Exon48.25.003GGAAACCTGAAAGGAAAATACATTT 524 Hu.DMD.Exon48.25.003.2CTCTGGAAACCTGAAAGGAAAATAC 525 Hu.DMD.Exon48.25.004GCTCTGGAAACCTGAAAGGAAAATA 526 Hu.DMD.Exon48.25.004.2TAAAGCTCTGGAAACCTGAAAGGAA 527 Hu.DMD.Exon48.25.005GTAAAGCTCTGGAAACCTGAAAGGA 528 Hu.DMD.Exon48.25.005.2TCAGGTAAAGCTCTGGAAACCTGAA 529 Hu.DMD.Exon48.25.006CTCAGGTAAAGCTCTGGAAACCTGA 530 Hu.DMD.Exon48.25.006.2GTTTCTCAGGTAAAGCTCTGGAAAC 531 Hu.DMD.Exon48.25.007TGTTTCTCAGGTAAAGCTCTGGAAA 532 Hu.DMD.Exon48.25.007.2AATTTCTCCTTGTTTCTCAGGTAAA 533 Hu.DMD.Exon48.25.008TTTGAGCTTCAATTTCTCCTTGTTT 534 Hu.DMD.Exon48.25.008TTTTATTTGAGCTTCAATTTCTCCT 535 Hu.DMD.Exon48.25.009AAGCTGCCCAAGGTCTTTTATTTGA 536 Hu.DMD.Exon48.25.010AGGTCTTCAAGCTTTTTTTCAAGCT 537 Hu.DMD.Exon48.25.010.2TTCAAGCTTTTTTTCAAGCTGCCCA 538 Hu.DMD.Exon48.25.011GATGATTTAACTGCTCTTCAAGGTC 539 Hu.DMD.Exon48.25.011.2CTGCTCTTCAAGGTCTTCAAGCTTT 540 Hu.DMD.Exon48.25.012AGGAGATAACCACAGCAGCAGATGA 541 Hu.DMD.Exon48.25.012.2CAGCAGATGATTTAACTGCTCTTCA 542 Hu.DMD.Exon48.25.013ATTTCCAACTGATTCCTAATAGGAG 543 Hu.DMD.Exon48.25.014CTTGGTTTGGTTGGTTATAAATTTC 544 Hu.DMD.Exon48.25.014.2CAACTGATTCCTAATAGGAGATAAC 545 Hu.DMD.Exon48.25.015CTTAACGTCAAATGGTCCTTCTTGG 546 Hu.DMD.Exon48.25.015.2TTGGTTATAAATTTCCAACTGATTC 547 Hu.DMD.Exon48.25.016CCTACCTTAACGTCAAATGGTCCTT 548 Hu.DMD.Exon48.25.016.2TCCTTCTTGGTTTGGTTGGTTATAA 549 Hu.DMD.Exon48.25.017AGTTCCCTACCTTAACGTCAAATGG 550 Hu.DMD.Exon48.25.018CAAAAAGTTCCCTACCTTAACGTCA 551 Hu.DMD.Exon48.25.019TAAAGCAAAAAGTTCCCTACCTTAA 552 Hu.DMD.Exon48.25.020ATATTTAAAGCAAAAAGTTCCCTAC 553 Hu.DMD.Exon48.20.001 AGGAAAATACATTTTAAAAA554 Hu.DMD.Exon48.20.002 AAGGAAAATACATTTTAAAA 555 Hu.DMD.Exon48.20.003CCTGAAAGGAAAATACATTT 556 Hu.DMD.Exon48.20.004 GGAAACCTGAAAGGAAAATA 557Hu.DMD.Exon48.20.005 GCTCTGGAAACCTGAAAGGA 558 Hu.DMD.Exon48.20.006GTAAAGCTCTGGAAACCTGA 559 Hu.DMD.Exon48.20.007 CTCAGGTAAAGCTCTGGAAA 560Hu.DMD.Exon48.20.008 AATTTCTCCTTGTTTCTCAG 561 Hu.DMD.Exon48.20.009TTTTATTTGAGCTTCAATTT 562 Hu.DMD.Exon48.20.010 AAGCTGCCCAAGGTCTTTTA 563Hu.DMD.Exon48.20.011 TTCAAGCTTTTTTTCAAGCT 564 Hu.DMD.Exon48.20.012CTGCTCTTCAAGGTCTTCAA 565 Hu.DMD.Exon48.20.013 CAGCAGATGATTTAACTGCT 566Hu.DMD.Exon48.20.014 AGGAGATAACCACAGCAGCA 567 Hu.DMD.Exon48.20.015CAACTGATTCCTAATAGGAG 568 Hu.DMD.Exon48.20.016 TTGGTTATAAATTTCCAACT 569Hu.DMD.Exon48.20.017 TCCTTCTTGGTTTGGTTGGT 570 Hu.DMD.Exon48.20.018CTTAACGTCAAATGGTCCTT 571 Hu.DMD.Exon48.20.019 CCTACCTTAACGTCAAATGG 572Hu.DMD.Exon48.20.020 AGTTCCCTACCTTAACGTCA 573 Hu.DMD.Exon48.20.021CAAAAAGTTCCCTACCTTAA 574 Hu.DMD.Exon48.20.022 TAAAGCAAAAAGTTCCCTAC 575Hu.DMD.Exon48.20.023 ATATTTAAAGCAAAAAGTTC 576 Hu.DMD.Exon49.25.001CTGGGGAAAAGAACCCATATAGTGC 577 Hu.DMD.Exon49.25.002TCCTGGGGAAAAGAACCCATATAGT 578 Hu.DMD.Exon49.25.002.2GTTTCCTGGGGAAAAGAACCCATAT 579 Hu.DMD.Exon49.25.003CAGTTTCCTGGGGAAAAGAACCCAT 580 Hu.DMD.Exon49.25.003.2TTTCAGTTTCCTGGGGAAAAGAACC 581 Hu.DMD.Exon49.25.004TATTTCAGTTTCCTGGGGAAAAGAA 582 Hu.DMD.Exon49.25.004.2TGCTATTTCAGTTTCCTGGGGAAAA 583 Hu.DMD.Exon49.25.005ACTGCTATTTCAGTTTCCTGGGGAA 584 Hu.DMD.Exon49.25.005.2TGAACTGCTATTTCAGTTTCCTGGG 585 Hu.DMD.Exon49.25.006CTTGAACTGCTATTTCAGTTTCCTG 586 Hu.DMD.Exon49.25.006.2TAGCTTGAACTGCTATTTCAGTTTC 587 Hu.DMD.Exon49.25.007TTTAGCTTGAACTGCTATTTCAGTT 588 Hu.DMD.Exon49.25.008TTCCACATCCGGTTGTTTAGCTTGA 589 Hu.DMD.Exon49.25.009TGCCCTTTAGACAAAATCTCTTCCA 590 Hu.DMD.Exon49.25.009.2TTTAGACAAAATCTCTTCCACATCC 591 Hu.DMD.Exon49.25.010GTTTTTCCTTGTACAAATGCTGCCC 592 Hu.DMD.Exon49.25.010.2GTACAAATGCTGCCCTTTAGACAAA 593 Hu.DMD.Exon49.25.011CTTCACTGGCTGAGTGGCTGGTTTT 594 Hu.DMD.Exon49.25.011.2GGCTGGTTTTTCCTTGTACAAATGC 595 Hu.DMD.Exon49.25.012ATTACCTTCACTGGCTGAGTGGCTG 596 Hu.DMD.Exon49.25.013GCTTCATTACCTTCACTGGCTGAGT 597 Hu.DMD.Exon49.25.014AGGTTGCTTCATTACCTTCACTGGC 598 Hu.DMD.Exon49.25.015GCTAGAGGTTGCTTCATTACCTTCA 599 Hu.DMD.Exon49.25.016ATATTGCTAGAGGTTGCTTCATTAC 600 Hu.DMD.Exon49.20.001 GAAAAGAACCCATATAGTGC601 Hu.DMD.Exon49.20.002 GGGAAAAGAACCCATATAGT 602 Hu.DMD.Exon49.20.003TCCTGGGGAAAAGAACCCAT 603 Hu.DMD.Exon49.20.004 CAGTTTCCTGGGGAAAAGAA 604Hu.DMD.Exon49.20.005 TATTTCAGTTTCCTGGGGAA 605 Hu.DMD.Exon49.20.006ACTGCTATTTCAGTTTCCTG 606 Hu.DMD.Exon49.20.007 CTTGAACTGCTATTTCAGTT 607Hu.DMD.Exon49.20.008 TTTAGCTTGAACTGCTATTT 608 Hu.DMD.Exon49.20.009TTCCACATCCGGTTGTTTAG 609 Hu.DMD.Exon49.20.010 TTTAGACAAAATCTCTTCCA 610Hu.DMD.Exon49.20.011 GTACAAATGCTGCCCTTTAG 611 Hu.DMD.Exon49.20.012GGCTGGTTTTTCCTTGTACA 612 Hu.DMD.Exon49.20.013 CTTCACTGGCTGAGTGGCTG 613Hu.DMD.Exon49.20.014 ATTACCTTCACTGGCTGAGT 614 Hu.DMD.Exon49.20.015GCTTCATTACCTTCACTGGC 615 Hu.DMD.Exon49.20.016 AGGTTGCTTCATTACCTTCA 616Hu.DMD.Exon49.20.017 GCTAGAGGTTGCTTCATTAC 617 Hu.DMD.Exon49.20.018ATATTGCTAGAGGTTGCTTC 618 Hu.DMD.Exon50.25.001 CTTTAACAGAAAAGCATACACATTA619 Hu.DMD.Exon50.25.002 TCCTCTTTAACAGAAAAGCATACAC 620Hu.DMD.Exon50.25.002.2 TTCCTCTTTAACAGAAAAGCATACA 621Hu.DMD.Exon50.25.003 TAACTTCCTCTTTAACAGAAAAGCA 622Hu.DMD.Exon50.25.003.2 CTAACTTCCTCTTTAACAGAAAAGC 623Hu.DMD.Exon50.25.004 TCTTCTAACTTCCTCTTTAACAGAA 624Hu.DMD.Exon50.25.004.2 ATCTTCTAACTTCCTCTTTAACAGA 625Hu.DMD.Exon50.25.005 TCAGATCTTCTAACTTCCTCTTTAA 626Hu.DMD.Exon50.25.005.2 CTCAGATCTTCTAACTTCCTCTTTA 627Hu.DMD.Exon50.25.006 AGAGCTCAGATCTTCTAACTTCCTC 628Hu.DMD.Exon50.25.006.2 CAGAGCTCAGATCTTCTAACTTCCT 629 NG-08-0731Hu.DMD.Exon50.25.007 CACTCAGAGCTCAGATCTTCTACT 630 Hu.DMD.Exon50.25.007.2CCTTCCACTCAGAGCTCAGATCTTC 631 Hu.DMD.Exon50.25.008GTAAACGGTTTACCGCCTTCCACTC 632 Hu.DMD.Exon50.25.009CTTTGCCCTCAGCTCTTGAAGTAAA 633 Hu.DMD.Exon50.25.009.2CCCTCAGCTCTTGAAGTAAACGGTT 634 Hu.DMD.Exon50.25.010CCAGGAGCTAGGTCAGGCTGCTTTG 635 Hu.DMD.Exon50.25.010.2GGTCAGGCTGCTTTGCCCTCAGCTC 636 Hu.DMD.Exon50.25.011AGGCTCCAATAGTGGTCAGTCCAGG 637 Hu.DMD.Exon50.25.011.2TCAGTCCAGGAGCTAGGTCAGGCTG 638 Hu.DMD.Exon50.25.012CTTACAGGCTCCAATAGTGGTCAGT 639 AVI-5038 Hu.DMD.Exon50.25.013GTATACTTACAGGCTCCAATAGTGG 640 Hu.DMD.Exon50.25.014ATCCAGTATACTTACAGGCTCCAAT 641 Hu.DMD.Exon50.25.015ATGGGATCCAGTATACTTACAGGCT 642 NG-08-0741 Hu.DMD.Exon50.25.016AGAGAATGGGATCCAGTATACTTAC 643 NG-08-0742 Hu.DMD.Exon50.20.001ACAGAAAAGCATACACATTA 644 Hu.DMD.Exon50.20.002 TTTAACAGAAAAGCATACAC 645Hu.DMD.Exon50.20.003 TCCTCTTTAACAGAAAAGCA 646 Hu.DMD.Exon50.20.004TAACTTCCTCTTTAACAGAA 647 Hu.DMD.Exon50.20.005 TCTTCTAACTTCCTCTTTAA 648Hu.DMD.Exon50.20.006 TCAGATCTTCTAACTTCCTC 649 Hu.DMD.Exon50.20.007CCTTCCACTCAGAGCTCAGA 650 Hu.DMD.Exon50.20.008 GTAAACGGTTTACCGCCTTC 651Hu.DMD.Exon50.20.009 CCCTCAGCTCTTGAAGTAAA 652 Hu.DMD.Exon50.20.010GGTCAGGCTGCTTTGCCCTC 653 Hu.DMD.Exon50.20.011 TCAGTCCAGGAGCTAGGTCA 654Hu.DMD.Exon50.20.012 AGGCTCCAATAGTGGTCAGT 655 Hu.DMD.Exon50.20.013CTTACAGGCTCCAATAGTGG 656 Hu.DMD.Exon50.20.014 GTATACTTACAGGCTCCAAT 657Hu.DMD.Exon50.20.015 ATCCAGTATACTTACAGGCT 658 Hu.DMD.Exon50.20.016ATGGGATCCAGTATACTTAC 659 Hu.DMD.Exon50.20.017 AGAGAATGGGATCCAGTATA 660Hu.DMD.Exon51.25.001-44 CTAAAATATTTTGGGTTTTTGCAAAA 661Hu.DMD.Exon51.25.002-45 GCTAAAATATTTTGGGTTTTTGCAAA 662Hu.DMD.Exon51.25.002.2-46 TAGGAGCTAAAATATTTTGGGTTTTT 663Hu.DMD.Exon51.25.003 AGTAGGAGCTAAAATATTTTGGGTT 664Hu.DMD.Exon51.25.003.2 TGAGTAGGAGCTAAAATATTTTGGG 665Hu.DMD.Exon51.25.004 CTGAGTAGGAGCTAAAATATTTTGGG 666Hu.DMD.Exon51.25.004.2 CAGTCTGAGTAGGAGCTAAAATATT 667Hu.DMD.Exon51.25.005 ACAGTCTGAGTAGGAGCTAAAATATT 668Hu.DMD.Exon51.25.005.2 GAGTAACAGTCTGAGTAGGAGCTAAA 669Hu.DMD.Exon51.25.006 CAGAGTAACAGTCTGAGTAGGAGCT 670Hu.DMD.Exon51.25.006.2 CACCAGAGTAACAGTCTGAGTAGGAG 671Hu.DMD.Exon51.25.007 GTCACCAGAGTAACAGTCTGAGTAG 672Hu.DMD.Exon51.25.007.2 AACCACAGGTTGTGTCACCAGAGTAA 673Hu.DMD.Exon51.25.008 GTTGTGTCACCAGAGTAACAGTCTG 674 Hu.DMD.Exon51.25.009TGGCAGTTTCCTTAGTAACCACAGGT 675 Hu.DMD.Exon51.25.010ATTTCTAGTTTGGAGATGGCAGTTTC 676 Hu.DMD.Exon51.25.010.2GGAAGATGGCATTTCTAGTTTGGAG 677 Hu.DMD.Exon51.25.011CATCAAGGAAGATGGCATTTCTAGTT 678 Hu.DMD.Exon51.25.011.2GAGCAGGTACCTCCAACATCAAGGAA 679 Hu.DMD.Exon51.25.012ATCTGCCAGAGCAGGTACCTCCAAC 680 Hu.DMD.Exon51.25.013AAGTTCTGTCCAAGCCCGGTTGAAAT 681 Hu.DMD.Exon51.25.013.2CGGTTGAAATCTGCCAGAGCAGGTAC 682 Hu.DMD.Exon51.25.014GAGAAAGCCAGTCGGTAAGTTCTGTC 683 Hu.DMD.Exon51.25.014.2GTCGGTAAGTTCTGTCCAAGCCCGG 684 Hu.DMD.Exon51.25.015ATAACTTGATCAAGCAGAGAAAGCCA 685 Hu.DMD.Exon51.25.015.2AAGCAGAGAAAGCCAGTCGGTAAGT 686 Hu.DMD.Exon51.25.016CACCCTCTGTGATTTTATAACTTGAT 687 Hu.DMD.Exon51.25.017CAAGGTCACCCACCATCACCCTCTGT 688 Hu.DMD.Exon51.25.017.2CATCACCCTCTGTGATTTTATAACT 689 Hu.DMD.Exon51.25.018CTTCTGCTTGATGATCATCTCGTTGA 690 Hu.DMD.Exon51.25.019CCTTCTGCTTGATGATCATCTCGTTG 691 Hu.DMD.Exon51.25.019.2ATCTCGTTGATATCCTCAAGGTCACC 692 Hu.DMD.Exon51.25.020TCATACCTTCTGCTTGATGATCATCT 693 Hu.DMD.Exon51.25.020.2TCATTTTTTCTCATACCTTCTGCTTG 694 Hu.DMD.Exon51.25.021TTTTCTCATACCTTCTGCTTGATGAT 695 Hu.DMD.Exon51.25.022TTTTATCATTTTTTCTCATACCTTCT 696 Hu.DMD.Exon51.25.023CCAACTTTTATCATTTTTTCTCATAC 697 Hu.DMD.Exon51.20.001 ATATTTTGGGTTTTTGCAAA698 Hu.DMD.Exon51.20.002 AAAATATTTTGGGTTTTTGC 699 Hu.DMD.Exon51.20.003GAGCTAAAATATTTTGGGTT 700 Hu.DMD.Exon51.20.004 AGTAGGAGCTAAAATATTTT 701Hu.DMD.Exon51.20.005 GTCTGAGTAGGAGCTAAAAT 702 Hu.DMD.Exon51.20.006TAACAGTCTGAGTAGGAGCT 703 Hu.DMD.Exon51.20.007 CAGAGTAACAGTCTGAGTAG 704Hu.DMD.Exon51.20.008 CACAGGTTGTGTCACCAGAG 705 Hu.DMD.Exon51.20.009AGTTTCCTTAGTAACCACAG 706 Hu.DMD.Exon51.20.010 TAGTTTGGAGATGGCAGTTT 707Hu.DMD.Exon51.20.011 GGAAGATGGCATTTCTAGTT 708 Hu.DMD.Exon51.20.012TACCTCCAACATCAAGGAAG 709 Hu.DMD.Exon51.20.013 ATCTGCCAGAGCAGGTACCT 710Hu.DMD.Exon51.20.014 CCAAGCCCGGTTGAAATCTG 711 Hu.DMD.Exon51.20.015GTCGGTAAGTTCTGTCCAAG 712 Hu.DMD.Exon51.20.016 AAGCAGAGAAAGCCAGTCGG 713Hu.DMD.Exon51.20.017 TTTTATAACTTGATCAAGCA 714 Hu.DMD.Exon51.20.018CATCACCCTCTGTGATTTTA 715 Hu.DMD.Exon51.20.019 CTCAAGGTCACCCACCATCA 716Hu.DMD.Exon51.20.020 CATCTCGTTGATATCCTCAA 717 Hu.DMD.Exon51.20.021CTTCTGCTTGATGATCATCT 718 Hu.DMD.Exon51.20.022 CATACCTTCTGCTTGATGAT 719Hu.DMD.Exon51.20.023 TTTCTCATACCTTCTGCTTG 720 Hu.DMD.Exon51.20.024CATTTTTTCTCATACCTTCT 721 Hu.DMD.Exon51.20.025 TTTATCATTTTTTCTCATAC 722Hu.DMD.Exon51.20.026 CAACTTTTATCATTTTTTCT 723 Hu.DMD.Exon52.25.001CTGTAAGAACAAATATCCCTTAGTA 724 Hu.DMD.Exon52.25.002TGCCTGTAAGAACAAATATCCCTTA 725 Hu.DMD.Exon52.25.002.2GTTGCCTGTAAGAACAAATATCCCT 726 Hu.DMD.Exon52.25.003ATTGTTGCCTGTAAGAACAAATATC 727 Hu.DMD.Exon52.25.003.2GCATTGTTGCCTGTAAGAACAAATA 728 Hu.DMD.Exon52.25.004CCTGCATTGTTGCCTGTAAGAACAA 729 Hu.DMD.Exon52.25.004.2ATCCTGCATTGTTGCCTGTAAGAAC 730 Hu.DMD.Exon52.25.005CAAATCCTGCATTGTTGCCTGTAAG 731 Hu.DMD.Exon52.25.005.2TCCAAATCCTGCATTGTTGCCTGTA 732 Hu.DMD.Exon52.25.006TGTTCCAAATCCTGCATTGTTGCCT 733 Hu.DMD.Exon52.25.006.2TCTGTTCCAAATCCTGCATTGTTGC 734 Hu.DMD.Exon52.25.007AACTGGGGACGCCTCTGTTCCAAAT 735 Hu.DMD.Exon52.25.007.2GCCTCTGTTCCAAATCCTGCATTGT 736 Hu.DMD.Exon52.25.008CAGCGGTAATGAGTTCTTCCAACTG 737 Hu.DMD.Exon52.25.008.2CTTCCAACTGGGGACGCCTCTGTTC 738 Hu.DMD.Exon52.25.009CTTGTTTTTCAAATTTTGGGCAGCG 739 Hu.DMD.Exon52.25.010CTAGCCTCTTGATTGCTGGTCTTGT 740 Hu.DMD.Exon52.25.010.2TTTTCAAATTTTGGGCAGCGGTAAT 741 Hu.DMD.Exon52.25.011TTCGATCCGTAATGATTGTTCTAGC 742 Hu.DMD.Exon52.25.011.2GATTGCTGGTCTTGTTTTTCAAATT 743 Hu.DMD.Exon52.25.012CTTACTTCGATCCGTAATGATTGTT 744 Hu.DMD.Exon52.25.012.2TTGTTCTAGCCTCTTGATTGCTGGT 745 Hu.DMD.Exon52.25.013AAAAACTTACTTCGATCCGTAATGA 746 Hu.DMD.Exon52.25.014TGTTAAAAAACTTACTTCGATCCGT 747 Hu.DMD.Exon52.25.015ATGCTTGTTAAAAAACTTACTTCGA 748 Hu.DMD.Exon52.25.016GTCCCATGCTTGTTAAAAAACTTAC 749 Hu.DMD.Exon52.20.001 AGAACAAATATCCCTTAGTA750 Hu.DMD.Exon52.20.002 GTAAGAACAAATATCCCTTA 751 Hu.DMD.Exon52.20.003TGCCTGTAAGAACAAATATC 752 Hu.DMD.Exon52.20.004 ATTGTTGCCTGTAAGAACAA 753Hu.DMD.Exon52.20.005 CCTGCATTGTTGCCTGTAAG 754 Hu.DMD.Exon52.20.006CAAATCCTGCATTGTTGCCT 755 Hu.DMD.Exon52.20.007 GCCTCTGTTCCAAATCCTGC 756Hu.DMD.Exon52.20.008 CTTCCAACTGGGGACGCCTC 757 Hu.DMD.Exon52.20.009CAGCGGTAATGAGTTCTTCC 758 Hu.DMD.Exon52.20.010 TTTTCAAATTTTGGGCAGCG 759Hu.DMD.Exon52.20.011 GATTGCTGGTCTTGTTTTTC 760 Hu.DMD.Exon52.20.012TTGTTCTAGCCTCTTGATTG 761 Hu.DMD.Exon52.20.013 TTCGATCCGTAATGATTGTT 762Hu.DMD.Exon52.20.014 CTTACTTCGATCCGTAATGA 763 Hu.DMD.Exon52.20.015AAAAACTTACTTCGATCCGT 764 Hu.DMD.Exon52.20.016 TGTTAAAAAACTTACTTCGA 765Hu.DMD.Exon52.20.017 ATGCTTGTTAAAAAACTTAC 766 Hu.DMD.Exon52.20.018GTCCCATGCTTGTTAAAAAA 767 Hu.DMD.Exon53.25.001 CTAGAATAAAAGGAAAAATAAATAT768 Hu.DMD.Exon53.25.002 AACTAGAATAAAAGGAAAAATAAAT 769Hu.DMD.Exon53.25.002.2 TTCAACTAGAATAAAAGGAAAAATA 770Hu.DMD.Exon53.25.003 CTTTCAACTAGAATAAAAGGAAAAA 771Hu.DMD.Exon53.25.003.2 ATTCTTTCAACTAGAATAAAAGGAA 772Hu.DMD.Exon53.25.004 GAATTCTTTCAACTAGAATAAAAGG 773Hu.DMD.Exon53.25.004.2 TCTGAATTCTTTCAACTAGAATAAA 774Hu.DMD.Exon53.25.005 ATTCTGAATTCTTTCAACTAGAATA 775Hu.DMD.Exon53.25.005.2 CTGATTCTGAATTCTTTCAACTAGA 776Hu.DMD.Exon53.25.006 CACTGATTCTGAATTCTTTCAACTA 777Hu.DMD.Exon53.25.006.2 TCCCACTGATTCTGAATTCTTTCAA 778Hu.DMD.Exon53.25.007 CATCCCACTGATTCTGAATTCTTTC 779 Hu.DMD.Exon53.25.008TACTTCATCCCACTGATTCTGAATT 780 Hu.DMD.Exon53.25.008.2CTGAAGGTGTTCTTGTACTTCATCC 781 Hu.DMD.Exon53.25.009CGGTTCTGAAGGTGTTCTTGTACT 782 Hu.DMD.Exon53.25.009.2CTGTTGCCTCCGGTTCTGAAGGTGT 783 Hu.DMD.Exon53.25.010TTTCATTCAACTGTTGCCTCCGGTT 784 Hu.DMD.Exon53.25.010.2TAACATTTCATTCAACTGTTGCCTC 785 Hu.DMD.Exon53.25.011TTGTGTTGAATCCTTTAACATTTCA 786 Hu.DMD.Exon53.25.012TCTTCCTTAGCTTCCAGCCATTGTG 787 Hu.DMD.Exon53.25.012.2CTTAGCTTCCAGCCATTGTGTTGAA 788 Hu.DMD.Exon53.25.013GTCCTAAGACCTGCTCAGCTTCTTC 789 Hu.DMD.Exon53.25.013.2CTGCTCAGCTTCTTCCTTAGCTTCC 790 Hu.DMD.Exon53.25.014CTCAAGCTTGGCTCTGGCCTGTCCT 791 Hu.DMD.Exon53.25.014.2GGCCTGTCCTAAGACCTGCTCAGCT 792 Hu.DMD.Exon53.25.015TAGGGACCCTCCTTCCATGACTCAA 793 Hu.DMD.Exon53.25.016TTTGGATTGCATCTACTGTATAGGG 794 Hu.DMD.Exon53.25.016.2ACCCTCCTTCCATGACTCAAGCTTG 795 Hu.DMD.Exon53.25.017CTTGGTTTCTGTGATTTTCTTTTGG 796 Hu.DMD.Exon53.25.017.2ATCTACTGTATAGGGACCCTCCTTC 797 Hu.DMD.Exon53.25.018CTAACCTTGGTTTCTGTGATTTTCT 798 Hu.DMD.Exon53.25.018.2TTTCTTTTGGATTGCATCTACTGTA 799 Hu.DMD.Exon53.25.019TGATACTAACCTTGGTTTCTGTGAT 800 Hu.DMD.Exon53.25.020ATCTTTGATACTAACCTTGGTTTCT 801 Hu.DMD.Exon53.25.021AAGGTATCTTTGATACTAACCTTGG 802 Hu.DMD.Exon53.25.022TTAAAAAGGTATCTTTGATACTAAC 803 Hu.DMD.Exon53.20.001 ATAAAAGGAAAAATAAATAT804 Hu.DMD.Exon53.20.002 GAATAAAAGGAAAAATAAAT 805 Hu.DMD.Exon53.20.003AACTAGAATAAAAGGAAAAA 806 Hu.DMD.Exon53.20.004 CTTTCAACTAGAATAAAAGG 807Hu.DMD.Exon53.20.005 GAATTCTTTCAACTAGAATA 808 Hu.DMD.Exon53.20.006ATTCTGAATTCTTTCAACTA 809 Hu.DMD.Exon53.20.007 TACTTCATCCCACTGATTCT 810Hu.DMD.Exon53.20.008 CTGAAGGTGTTCTTGTACT 811 Hu.DMD.Exon53.20.009CTGTTGCCTCCGGTTCTGAA 812 Hu.DMD.Exon53.20.010 TAACATTTCATTCAACTGTT 813Hu.DMD.Exon53.20.011 TTGTGTTGAATCCTTTAACA 814 Hu.DMD.Exon53.20.012CTTAGCTTCCAGCCATTGTG 815 Hu.DMD.Exon53.20.013 CTGCTCAGCTTCTTCCTTAG 816Hu.DMD.Exon53.20.014 GGCCTGTCCTAAGACCTGCT 817 Hu.DMD.Exon53.20.015CTCAAGCTTGGCTCTGGCCT 818 Hu.DMD.Exon53.20.016 ACCCTCCTTCCATGACTCAA 819Hu.DMD.Exon53.20.017 ATCTACTGTATAGGGACCCT 820 Hu.DMD.Exon53.20.018TTTCTTTTGGATTGCATCTA 821 Hu.DMD.Exon53.20.019 CTTGGTTTCTGTGATTTTCT 822Hu.DMD.Exon53.20.020 CTAACCTTGGTTTCTGTGAT 823 Hu.DMD.Exon53.20.021TGATACTAACCTTGGTTTCT 824 Hu.DMD.Exon53.20.022 ATCTTTGATACTAACCTTGG 825Hu.DMD.Exon53.20.023 AAGGTATCTTTGATACTAAC 826 Hu.DMD.Exon53.20.024TTAAAAAGGTATCTTTGATA 827 Hu.DMD.Exon54.25.001 CTATAGATTTTTATGAGAAAGAGA828 Hu.DMD.Exon54.25.002 AACTGCTATAGATTTTTATGAGAAA 829Hu.DMD.Exon54.25.003 TGGCCAACTGCTATAGATTTTTATG 830 Hu.DMD.Exon54.25.004GTCTTTGGCCAACTGCTATAGATTT 831 Hu.DMD.Exon54.25.005CGGAGGTCTTTGGCCAACTGCTATA 832 Hu.DMD.Exon54.25.006ACTGGCGGAGGTCTTTGGCCAACTG 833 Hu.DMD.Exon54.25.007TTTGTCTGCCACTGGCGGAGGTCTT 834 Hu.DMD.Exon54.25.008AGTCATTTGCCACATCTACATTTGT 835 Hu.DMD.Exon54.25.008.2TTTGCCACATCTACATTTGTCTGCC 836 Hu.DMD.Exon54.25.009CCGGAGAAGTTTCAGGGCCAAGTCA 837 Hu.DMD.Exon54.25.010GTATCATCTGCAGAATAATCCCGGA 838 Hu.DMD.Exon54.25.010.2TAATCCCGGAGAAGTTTCAGGGCCA 839 Hu.DMD.Exon54.25.011TTATCATGTGGACTTTTCTGGTATC 840 Hu.DMD.Exon54.25.012AGAGGCATTGATATTCTCTGTTATC 841 Hu.DMD.Exon54.25.012.2ATGTGGACTTTTCTGGTATCATCTG 842 Hu.DMD.Exon54.25.013CTTTTATGAATGCTTCTCCAAGAGG 843 Hu.DMD.Exon54.25.013.2ATATTCTCTGTTATCATGTGGACTT 844 Hu.DMD.Exon54.25.014CATACCTTTTATGAATGCTTCTCCA 845 Hu.DMD.Exon54.25.014.2CTCCAAGAGGCATTGATATTCTCTG 846 Hu.DMD.Exon54.25.015TAATTCATACCTTTTATGAATGCTT 847 Hu.DMD.Exon54.25.015.2CTTTTATGAATGCTTCTCCAAGAGG 848 Hu.DMD.Exon54.25.016TAATGTAATTCATACCTTTTATGAA 849 Hu.DMD.Exon54.25.017AGAAATAATGTAATTCATACCTTTT 850 Hu.DMD.Exon54.25.018GTTTTAGAAATAATGTAATTCATAC 851 Hu.DMD.Exon54.20.001 GATTTTTATGAGAAAGAGA852 Hu.DMD.Exon54.20.002 CTATAGATTTTTATGAGAAA 853 Hu.DMD.Exon54.20.003AACTGCTATAGATTTTTATG 854 Hu.DMD.Exon54.20.004 TGGCCAACTGCTATAGATTT 855Hu.DMD.Exon54.20.005 GTCTTTGGCCAACTGCTATA 856 Hu.DMD.Exon54.20.006CGGAGGTCTTTGGCCAACTG 857 Hu.DMD.Exon54.20.007 TTTGTCTGCCACTGGCGGAG 858Hu.DMD.Exon54.20.008 TTTGCCACATCTACATTTGT 859 Hu.DMD.Exon54.20.009TTCAGGGCCAAGTCATTTGC 860 Hu.DMD.Exon54.20.010 TAATCCCGGAGAAGTTTCAG 861Hu.DMD.Exon54.20.011 GTATCATCTGCAGAATAATC 862 Hu.DMD.Exon54.20.012ATGTGGACTTTTCTGGTATC 863 Hu.DMD.Exon54.20.013 ATATTCTCTGTTATCATGTG 864Hu.DMD.Exon54.20.014 CTCCAAGAGGCATTGATATT 865 Hu.DMD.Exon54.20.015CTTTTATGAATGCTTCTCCA 866 Hu.DMD.Exon54.20.016 CATACCTTTTATGAATGCTT 867Hu.DMD.Exon54.20.017 TAATTCATACCTTTTATGAA 868 Hu.DMD.Exon54.20.018TAATGTAATTCATACCTTTT 869 Hu.DMD.Exon54.20.019 AGAAATAATGTAATTCATAC 870Hu.DMD.Exon54.20.020 GTTTTAGAAATAATGTAATT 871 Hu.DMD.Exon55.25.001CTGCAAAGGACCAAATGTTCAGATG 872 Hu.DMD.Exon55.25.002TCACCCTGCAAAGGACCAAATGTTC 873 Hu.DMD.Exon55.25.003CTCACTCACCCTGCAAAGGACCAAA 874 Hu.DMD.Exon55.25.004TCTCGCTCACTCACCCTGCAAAGGA 875 Hu.DMD.Exon55.25.005CAGCCTCTCGCTCACTCACCCTGCA 876 Hu.DMD.Exon55.25.006CAAAGCAGCCTCTCGCTCACTCACC 877 Hu.DMD.Exon55.25.007TCTTCCAAAGCAGCCTCTCGCTCAC 878 Hu.DMD.Exon55.25.007.2TCTATGAGTTTCTTCCAAAGCAGCC 879 Hu.DMD.Exon55.25.008GTTGCAGTAATCTATGAGTTTCTTC 880 Hu.DMD.Exon55.25.008.2GAACTGTTGCAGTAATCTATGAGTT 881 Hu.DMD.Exon55.25.009TTCCAGGTCCAGGGGGAACTGTTGC 882 Hu.DMD.Exon55.25.010GTAAGCCAGGCAAGAAACTTTTCCA 883 Hu.DMD.Exon55.25.010.2CCAGGCAAGAAACTTTTCCAGGTCC 884 Hu.DMD.Exon55.25.011TGGCAGTTGTTTCAGCTTCTGTAAG 885 Hu.DMD.Exon55.25.011.2TTCAGCTTCTGTAAGCCAGGCAAGA 886 Hu.DMD.Exon55.25.012GGTAGCATCCTGTAGGACATTGGCA 887 Hu.DMD.Exon55.25.012.2GACATTGGCAGTTGTTTCAGCTTCT 888 Hu.DMD.Exon55.25.013TCTAGGAGCCTTTCCTTACGGGTAG 889 Hu.DMD.Exon55.25.014CTTTTACTCCCTTGGAGTCTTCTAG 890 Hu.DMD.Exon55.25.014.2GAGCCTTTCCTTACGGGTAGCATCC 891 Hu.DMD.Exon55.25.015TTGCCATTGTTTCATCAGCTCTTTT 892 Hu.DMD.Exon55.25.015.2CTTGGAGTCTTCTAGGAGCCTTTCC 893 Hu.DMD.Exon55.25.016CTTACTTGCCATTGTTTCATCAGCT 894 Hu.DMD.Exon55.25.016.2CAGCTCTTTTACTCCCTTGGAGTCT 895 Hu.DMD.Exon55.25.017CCTGACTTACTTGCCATTGTTTCAT 896 Hu.DMD.Exon55.25.018AAATGCCTGACTTACTTGCCATTGT 897 Hu.DMD.Exon55.25.019AGCGGAAATGCCTGACTTACTTGCC 898 Hu.DMD.Exon55.25.020GCTAAAGCGGAAATGCCTGACTTAC 899 Hu.DMD.Exon55.20.001 AAGGACCAAATGTTCAGATG900 Hu.DMD.Exon55.20.002 CTGCAAAGGACCAAATGTTC 901 Hu.DMD.Exon55.20.003TCACCCTGCAAAGGACCAAA 902 Hu.DMD.Exon55.20.004 CTCACTCACCCTGCAAAGGA 903Hu.DMD.Exon55.20.005 TCTCGCTCACTCACCCTGCA 904 Hu.DMD.Exon55.20.006CAGCCTCTCGCTCACTCACC 905 Hu.DMD.Exon55.20.007 CAAAGCAGCCTCTCGCTCAC 906Hu.DMD.Exon55.20.008 TCTATGAGTTTCTTCCAAAG 907 Hu.DMD.Exon55.20.009GAACTGTTGCAGTAATCTAT 908 Hu.DMD.Exon55.20.010 TTCCAGGTCCAGGGGGAACT 909Hu.DMD.Exon55.20.011 CCAGGCAAGAAACTTTTCCA 910 Hu.DMD.Exon55.20.012TTCAGCTTCTGTAAGCCAGG 911 Hu.DMD.Exon55.20.013 GACATTGGCAGTTGTTTCAG 912Hu.DMD.Exon55.20.014 GGTAGCATCCTGTAGGACAT 913 Hu.DMD.Exon55.20.015GAGCCTTTCCTTACGGGTAG 914 Hu.DMD.Exon55.20.016 CTTGGAGTCTTCTAGGAGCC 915Hu.DMD.Exon55.20.017 CAGCTCTTTTACTCCCTTGG 916 Hu.DMD.Exon55.20.018TTGCCATTGTTTCATCAGCT 917 Hu.DMD.Exon55.20.019 CTTACTTGCCATTGTTTCAT 918Hu.DMD.Exon55.20.020 CCTGACTTACTTGCCATTGT 919 Hu.DMD.Exon55.20.021AAATGCCTGACTTACTTGCC 920 Hu.DMD.Exon55.20.022 AGCGGAAATGCCTGACTTAC 921Hu.DMD.Exon55.20.023 GCTAAAGCGGAAATGCCTGA 922 H50A(+02+30)-AVI-5656CCACTCAGAGCTCAGATCTTCTAACTTCC 923 H5OD(+07-18)-AVI-5915GGGATCCAGTATACTTACAGGCTCC 924 H50A(+07+33) CTTCCACTCAGAGCTCAGATCTTCTAA925 H51A(+61+90)-AVI-4657 ACATCAAGGAAGATGGCATTTCTAGTTTGG 926H51A(+66+95)-AVI-4658 CTCCAACATCAAGGAAGATGGCATTTCTAG 927 H51A(+111+134)TTCTGTCCAAGCCCGGTTGAAATC 928 H51A(+175+195) CACCCACCATCACCCTCYGTG 929H51A(+199+220) ATCATCTCGTTGATATCCTCAA 930 H51A(+66+90)ACATCAAGGAAGATGGCATTTCTAG 931 H51A(-01+25) ACCAGAGTAACAGTCTGAGTAGGAGC932 h51AON1 TCAAGGAAGATGGCATTTCT 933 h51AON2 CCTCTGTGATTTTATAACTTGAT 934H51D(+08-17) ATCATTTTTTCTCATACCTTCTGCT 935 H51D(+16-07)CTCATACCTTCTGCTTGATGATC 936 hAON#23 TGGCATTTCTAGTTTGG 937 hAON#24CCAGAGCAGGTACCTCCAACATC 938 H44A(+61+84) TGTTCAGCTTCTGTTAGCCACTGA 939H44A(+85+104) TTTGTGTCTTTCTGAGAAAC 940 h44AON1 CGCCGCCATTTCTCAACAG 941H44A(-06+14) ATCTGTCAAATCGCCTGCAG 942 H45A(+71+90) TGTTTTTGAGGATTGCTGAA943 h45AON1 GCTGAATTATTTCTTCCCC 944 h45AON5 GCCCAATGCCATCCTGG 945H45A(-06+20) CCAATGCCATCCTGGAGTTCCTGTAA 946 H53A(+39+69)CATTCAACTGTTGCCTCCGGTTCTGAAGGTG 947 H53A(+23+47)CTGAAGGTGTTCTTGTACTTCATCC 948 h53AON1 CTGTTGCCTCCGGTTCTG 949H53A(-12+10) ATTCTTTCAACTAGAATAAAAG 950 huEx45.30.66GCCATCCTGGAGTTCCTGTAAGATACCAAA 951 huEx45.30.71CCAATGCCATCCTGGAGTTCCTGTAAGATA 952 huEx45.30.79GCCGCTGCCCAATGCCATCCTGGAGTTCCT 953 huEx45.30.83GTTTGCCGCTGCCCAATGCCATCCTGGAGT 954 huEx45.30.88CAACAGTTTGCCGCTGCCCAATGCCATCCT 955 huEx45.30.92CTGACAACAGTTTGCCGCTGCCCAATGCCA 956 huEx45.30.96TGTTCTGACAACAGTTTGCCGCTGCCCAAT 957 huEx45.30.99CAATGTTCTGACAACAGTTTGCCGCTGCCC 958 huEx45.30.103CATTCAATGTTCTGACAACAGTTTGCCGCT 959 huEx45.30.120TATTTCTTCCCCAGTTGCATTCAATGTTCT 960 huEx45.30.127GCTGAATTATTTCTTCCCCAGTTGCATTCA 961 huEx45.30.132GGATTGCTGAATTATTTCTTCCCCAGTTGC 962 huEx45.30.137TTTGAGGATTGCTGAATTATTTCTTCCCCA 963 huEx53.30.84GTACTTCATCCCACTGATTCTGAATTCTTT 964 huEx53.30.88TCTTGTACTTCATCCCACTGATTCTGAATT 965 huEx53.30.91TGTTCTTGTACTTCATCCCACTGATTCTGA 966 huEx53.30.103CGGTTCTGAAGGTGTTCTTGTACTTCATCC 967 huEx53.30.106CTCCGGTTCTGAAGGTGTTCTTGTACTTCA 968 huEx53.30.109TGCCTCCGGTTCTGAAGGTGTTCTTGTACT 969 huEx53.30.112TGTTGCCTCCGGTTCTGAAGGTGTTCTTGT 970 huEx53.30.115AACTGTTGCCTCCGGTTCTGAAGGTGTTCT 971 huEx53.30.118TTCAACTGTTGCCTCCGGTTCTGAAGGTGT 972

Step 1: Antibody Conjugation with Maleimide-PEG-NHS Followed bysiRNA-DMD Conjugates

Anti-dystrophin antibody is exchanged with 1× Phosphate buffer (pH 7.4)and made up to 5 mg/mi concentration. To this solution, 2 equivalents ofSMCC linker or maleimide-PEGxkDa-NHS (x=1, 5, 10, 20) is added androtated for 4 hours at room temperature. Unreacted maleimide-PEG isremoved by spin filtration using 50 kDa MWCO Amicon spin filters and PBSpH 7.4. The antibody-PEG-Mal conjugate is collected and transferred intoa reaction vessel. Various siRNA conjugates are synthesized usingsequences listed in Tables 13-17. siRNA-DMD conjugates (2 equivalents)is added at RT to the antibody-PEG-maleimide in PBS and rotatedovernight. The reaction mixture is analyzed by analytical SAX columnchromatography and conjugate along with Unreacted antibody and siRNA isseen.

Step 2: Purification

The crude reaction mixture is purified by AKTA explorer FPLC using anionexchange chromatography. Fractions containing the antibody-PEG-DMDconjugate are pooled, concentrated and buffer exchanged with PBS, pH7.4. Antibody siRNA conjugates with SMCC linker, PEG1 kDa, PEG5 kDa andPEG10 kDa are separated based on the siRNA loading.

Step-3: Analysis of the Purified Conjugate

The isolated conjugate is characterized by either mass spec or SDS-PAGE.The purity of the conjugate is assessed by analytical HPLC using anionexchange chromatography.

The examples and embodiments described herein are for illustrativepurposes only and various modifications or changes suggested to personsskilled in the art are to be included within the spirit and purview ofthis application and scope of the appended claims.

What is claimed is:
 1. A single stranded oligonucleotide conjugate comprising an anti-transferrin receptor antibody or antigen binding fragment thereof conjugated to a single stranded oligonucleotide hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript of the DMD gene; wherein the single stranded oligonucleotide induces exon skipping in said pre-mRNA transcript to generate a mRNA transcript encoding a truncated DMD protein.
 2. The single stranded oligonucleotide conjugate of claim 1, wherein the single stranded oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO) or an antisense oligonucleotide (ASO).
 3. The single stranded oligonucleotide conjugate of claim 1, wherein the single stranded oligonucleotide is delivered into a muscle cell.
 4. The single stranded oligonucleotide conjugate of claim 1, wherein the single stranded oligonucleotide induces skipping of exon 8 of the DMD gene.
 5. The single stranded oligonucleotide conjugate of claim 1, wherein the single stranded oligonucleotide induces skipping of exon 23 of the DMD gene.
 6. The single stranded oligonucleotide conjugate of claim 1, wherein the single stranded oligonucleotide induces skipping of exon 35 of the DMD gene.
 7. The single stranded oligonucleotide conjugate of claim 1, wherein the single stranded oligonucleotide induces skipping of exon 43 of the DMD gene.
 8. The single stranded oligonucleotide conjugate of claim 1, wherein the single stranded oligonucleotide induces skipping of exon 44 of the DMD gene.
 9. The single stranded oligonucleotide conjugate of claim 1, wherein the single stranded oligonucleotide induces skipping of exon 45 of the DMD gene.
 10. The single stranded oligonucleotide conjugate of claim 1, wherein the single stranded oligonucleotide induces skipping of exon 50 of the DMD gene.
 11. The single stranded oligonucleotide conjugate of claim 1, wherein the single stranded oligonucleotide induces skipping of exon 51 of the DMD gene.
 12. The single stranded oligonucleotide conjugate of claim 1, wherein the single stranded oligonucleotide induces skipping of exon 52 of the DMD gene.
 13. The single stranded oligonucleotide conjugate of claim 1, wherein the single stranded oligonucleotide induces skipping of exon 53 of the DMD gene.
 14. The single stranded oligonucleotide conjugate of claim 1, wherein the single stranded oligonucleotide induces skipping of exon 55 of the DMD gene.
 15. The single stranded oligonucleotide conjugate of claim 1, wherein the antibody or antigen binding fragment thereof comprises a humanized antibody or antigen binding fragment thereof, chimeric antibody or antigen binding fragment thereof, monoclonal antibody or antigen binding fragment thereof, monovalent Fab′, divalent Fab2, single chain variable fragment (scFv), diabody, minibody, nanobody, single domain antibody (sdAb), or camelid antibody or antigen binding fragment thereof.
 16. The single stranded oligonucleotide conjugate of claim 1, wherein the single stranded oligonucleotide comprises at least from about 10 to about 30 nucleotides in length.
 17. The single stranded oligonucleotide conjugate of claim 1, wherein the single stranded oligonucleotide is conjugated to the antibody or antigen binding fragment thereof via a linker.
 18. The single stranded oligonucleotide conjugate of claim 17, wherein the linker is a cleavable linker.
 19. The single stranded oligonucleotide conjugate of claim 17, wherein the linker is a non-cleavable linker.
 20. The single stranded oligonucleotide conjugate of claim 17, wherein the linker is selected from the group consisting of a heterobifunctional linker, a homobifunctional linker, a maleimide group, a dipeptide moiety, a benzoic acid group or derivatives thereof, a C₁-C₆ alkyl A group, and a combination thereof.
 21. The single stranded oligonucleotide conjugate of claim 1, wherein the single stranded oligonucleotide conjugate has a single stranded oligonucleotide to antibody ratio of about 1:1, 2:1, 3:1, or 4:1.
 22. The single stranded oligonucleotide conjugate of claim 1, wherein the single stranded oligonucleotide conjugate is formulated for parenteral administration.
 23. The single stranded oligonucleotide conjugate of claim 1, wherein the truncated DMD protein modulates muscular dystrophy.
 24. The single stranded oligonucleotide of claim 23, wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. 